Methods for the synthesis of olefins and derivatives

ABSTRACT

The invention provides a method of producing acrylic acid. The method includes contacting fumaric acid with a sufficient amount of ethylene in the presence of a cross-metathesis transformation catalyst to produce about two moles of acrylic acid per mole of fumaric acid. Also provided is an acrylate ester. The method includes contacting fumarate diester with a sufficient amount of ethylene in the presence of a cross-metathesis transformation catalyst to produce about two moles of acrylate ester per mole of fumarate diester. An integrated process for process for producing acrylic acid or acrylate ester is provided which couples bioproduction of fumaric acid with metathesis transformation. An acrylic acid and an acrylate ester production also is provided.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority under 35 U.S.C. § 119(e) to U.S.Provisional Application No. 60/955,321 which is incorporated byreference herein in its entirety.

STATEMENT OF FEDERALLY SPONSORED RESEARCH

This invention was made with government support under grant numberDE-FG02-06ER84536 and DE-FG02-07ER84865 awarded by the Department ofEnergy. The United States Government has certain rights in thisinvention.

BACKGROUND OF THE INVENTION

This invention relates generally to the production of commodity andspecialty chemicals and, more specifically to an integrated bioprocessfor producing acrylic acid and acrylate esters.

Acrylic acid and acrylate esters are large volume petrochemicalproducts. For example, acrylic acid is a commodity monomer intermediateused for the production of polymeric materials such polyacrylic acid,which is a major component of superabsorbant diapers. Acrylic acid alsois used for the production of acrylate esters, which are utilized inwater-soluble latex coatings, adhesives and inks. Acrylic acid andacrylate esters are manufactured by petrochemical processes such asoxidation of propylene, followed by esterification with alcohols such asmethanol, butanol, and 2-ethylhexanol. These chemical products aremanufactured at total volumes exceeding 10 billion lb/year and representa market of over $10 B in sales. The annual growth for these markets isestimated to be 4-5% globally.

Chemicals manufactured from petroleum feedstocks suffer the burden ofhigh and volatile prices, insecure foreign supply chains, and decliningreserves (Frost, J. W., Redefining chemical manufacture. Ind.Biotechnol. 1:23-24 (2005)). Therefore, a method of producing largevolume chemicals or their intermediates by alternative means that reducepetroleum-based processes and also use less energy- andcapital-intensive processes would be beneficial. The ability to generatechemical compounds based on biological processes could provide one suchalternative means. However, complete biosynthesis of a chemical compoundis not always available, and in some instances, toxic to the hostorganism.

Chemical manufacture based on low cost renewable resources is anotheralternative for chemical manufacture as a possible displacement ofpetroleum-based raw materials such as propylene or butane. However, inorder for such resources to replace current manufacturing methods newchemical or biosynthetic processes need to be developed for eachresource and/or target chemical.

Thus, there exists a need for compositions and methods that reduce theuse for petroleum-based synthesis of acrylic acid and its derivatives.The present invention satisfies this need and provides relatedadvantages as well.

SUMMARY OF THE INVENTION

The invention provides a method of producing acrylic acid. The methodincludes contacting fumaric acid with a sufficient amount of ethylene inthe presence of a cross-metathesis transformation catalyst to produceabout two moles of acrylic acid per mole of fumaric acid. Also providedis an acrylate ester. The method includes contacting fumarate diesterwith a sufficient amount of ethylene in the presence of across-metathesis transformation catalyst to produce about two moles ofacrylate ester per mole of fumarate diester. An integrated process forprocess for producing acrylic acid or acrylate ester is provided whichcouples bioproduction of fumaric acid with metathesis transformation. Anacrylic acid and an acrylate ester production also is provided.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic diagram showing the synthesis of acrylic acidthrough cross-metathesis between fumaric acid and ethylene (Scheme 1)

FIG. 2 is a schematic diagram showing the synthesis of acrylate esterthrough cross-metathesis between fumarate diester and ethylene (Scheme2)

FIG. 3 is a schematic diagram showing an integrated bioproduction systemfor acrylic acid from glucose through biosynthesis of fumaric acid.

FIG. 4 is a bar graph showing the yield of ethyl acrylate as a functionmetathesis catalyst.

DETAILED DESCRIPTION OF THE INVENTION

This invention is directed to a method of synthesis for acrylic acid andits derivatives. The method provides an efficient process for productionof two moles of acrylic acid product per mole of fumaric acid reactant.The chemical synthesis method of the invention can be coupled withbioproduction of fumaric acid or fumarate ester for efficientutilization of carbon where one mole of a carbon source such as glucosecan yield up to four moles of acrylic acid. Another particularly usefuloutcome of coupling a chemical synthesis step with bioproduction of areactant intermediate is that it avoids possible toxic effects onproduction organisms that could result from the complete biosynthesis ofacrylic acid or acrylate esters.

In one specific embodiment, the invention is directed to the chemicalsynthesis of acrylic acid or acrylate ester from fumaric acid orfumarate diester. The method utilizes cross-metathesis transformation toexchange double bonds between fumaric acid and ethylene, resulting intwo moles of acrylic acid per mole of fumaric acid. With respect toacrylate ester formation, a cross-metathesis transformation is used toconvert one mole of fumarate diester to two moles of acrylate ester. Theester group can include a wide range of different chemical moieties.

In another specific embodiment, the invention is directed to a processthat couples a fumaric acid producing microbial organism with thechemical synthesis of acrylic acid or acrylate ester. The fumaric acidproducing microbial organism contains a set of metabolic modificationsthat necessarily couple fumaric acid production to growth. Fumaric acidin the culture medium or fermentation broth can be converted directly toacrylic acid by cross-metathesis with ethylene, or first isolated withsubsequent transformation. Acrylate esters are produced followingdiesterification of the biosynthesized fumaric acid.

As used herein, the term “acrylic acid” is intended to mean thecarboxylic acid having the chemical formula C₃H₄O₂, a molecular mass of72.06 g/mol with a melting point of 12° C. and a boiling point of 139°C. Acrylic acid is a clear, colorless liquid that is soluble, forexample, in water and fully miscible in, for example, alcohols, ethersand chloroform. Acrylic acid is the simplest unsaturated carboxylic acidwith both a double bond and a carboxyl group. Acrylic acid also is knownin the art as 2-propenoic acid, propenoic acid, acroleic acid,ethylenecarboxylic acid, propene acid and vinylformic acid. The term isintended to include the acrylate ion and salt forms of acrylic acid.

As used herein, the term “acrylate ester” is intended to mean the esterform of acrylic acid. An ester is represented by the general chemicalformula RCO₂R′ where R and R′ can be the same or different, and can beeither aliphatic or aromatic and wherein the aliphatic or aromaticmoiety can be substituted or unsubstituted. For an acrylate ester, Rcorresponds to the ethyenyl (CH2═CH) moiety of the ester.

As used herein, the term “fumaric acid” is intended to mean thedicarboxylic acid having the chemical formula C₄H₄O₄, a molecular massof 116.07 g/mol with a melting point of 287° C. and a white solidappearance. Fumaric acid is soluble, for example, in water and alcoholsand is generally known to be a precursor to L-malate in the Krebs cycleand in various fermentation processes. Fumaric acid also is known in theart as (E)-butenedioic acid, trans-1,2-ethylenedicarboxylic acid,2-butenedioic acid, allomaleic acid, boletic acid and lichenic acid. Theterm is intended to include the fumarate ion and salt forms of fumaricacid.

As used herein, the term “fumarate ester” is intended to mean an esterform of fumaric acid where R in the general chemical formula RCO₂R′corresponds to the fumaric acid moiety and R′ can be the same ordifferent, and can be either aliphatic or aromatic and wherein thealiphatic or aromatic moiety can be substituted or unsubstituted.Because fumaric acid is a dicarboxylic acid a fumarate ester can includea R′ moiety at either or both carboxyl groups. A fumarate ester havingboth carboxyl groups condensed into an ester is referred to herein as a“fumarate diester” and can be represented by the general formulaRlO₂CRCO₂R2, where R corresponds to the ethenylene (CH═CH) moiety and R,R1 and R2 can be can be the same or different and can be eitheraliphatic or aromatic.

As used herein, the term “ethylene” is intended to mean the chemicalcompound having the formula C₂H₄, a molecular mass of 28.05 g/mol with amelting point of ⁻169.1° C. and a boiling point of ⁻103.7° C. Ethyleneis a colorless flammable gas that exhibits solubility in water. Ethylenealso is known in the art as ethene.

As used herein, the term “metathesis transformation,” “cross-metathesistransformation” or a grammatically equivalent form thereof, is intendedto mean a bimolecular process formally involving the exchange of a bondor bonds between similar interacting chemical species so that thebonding affiliations in the products are substantially the same orsubstantially similar to those in the reactants. A metathesistransformation can be schematically represented by the general reaction:RCH═CHR+R′CH═CHR′→RCH═R′CH+RCH═R′CH. When used in reference to chemicalconversion of fumaric acid or a fumarate ester or diester to acrylicacid, acrylate ester or 2 acrylate esters, respectively, the term isintended to mean the exchange of double bonds between fumaric acid,fumarate ester or fumarate diester and an alkene group. Metathesistransformations are well known in the art and can be found described in,for example, Grubbs, R. H. Olefin Metathesis. Tetrahedron 60:7117-40(2004), and R. H. Grubbs, Handbook of Metathesis, Wiley-VCH, New York,2003.

As used herein, the term “diesterification” is intended to mean anesterification reaction of a dicarboxylic acid to form a diester. Anesterification reaction refers to a condensation reaction in which twomolecules or moieties unite to form a single molecule with the loss of asmall molecule such as water, hydrogen chloride, methanol or aceticacid, for example. Accordingly, diesterification of a fumaric acid ofthe invention condenses fumaric acid and an alcohol, for example, toform fumarate diester with the elimination of water.

A specific example of an esterification reaction include Fisheresterification, which refers to the process of forming an ester byrefluxing a carboxylic acid and an alcohol in the presence of an acidcatalyst. Catalysts well known in the art for Fisher esterificationinclude, for example, sulfuric acid, p-toluene sulfonic acid and Lewisacids such as scandium(III) triflate. General reaction times can varyfrom about 1-10 hours at temperatures of 60-110° C. Esterificationreactions are well known to those skilled in the art. Esterificationreactions well known in the art other than Fisher esterification alsocan be used in an esterification reaction of the invention, such asreaction between a carboxylic acid chloride and an alcohol in thepresence of a base such as pyridine, a tertiary amine, or aqueous sodiumhydroxide. The last procedure is referred to commonly as theSchotten-Baumann reaction. Esterification reactions includingmechanisms, substrates, reagents and conditions can be found describedin, for example, Morrison and Boyd, Organic Chemisty, Sixth Edition,Prentice Hall, N.J. (1992); Carey, F. A. and Sundberg, R. J., AdvancedOrganic Chemistry, Parts A and B, Third Edition, Plenum Press, New York(1990), and March's Advanced Organic Chemistry, 5th edition, 2001.

The term “esterification reagent” as it is used herein is intended tomean a chemical that is suitable for use in an esterification reaction.Therefore, esterification reagents include reactants such as acarboxylic acid and/or an alcohol as well as a catalyst or otherchemically reactive compound that can be included in the chemicalreaction. An esterification reagent also includes a diesterificationreagent when used with a dicarboxylic acid. For example, the chemistryat one carboxyl group of the dicarboxylic acid fumaric acid of theinvention is substantially the same as the chemistry at its secondcarboxyl group. Similarly, an esterification reagent also includesreagents that can react and form esters with more than two carboxylgroups on the same substrate. An example of a reactive compound isdicyclohexycarbodiimide, which acts as a dehydrating agent andfacilitates esterification processes through formation ofdicyclohexylurea.

As used herein, the term “catalyst” is intended to mean a substance thatincreases the rate of a chemical reaction without a net change in theamount of that substance in the system. Therefore, when used inreference to a cross-metathesis transformation the term is intended torefer to a substance that increases the rate of the bimolecular exchangeof bonds but is not consumed in the transformation. A specific exampleof a class of metathesis transformation catalysts is the rutheniummetathesis catalysts which are described in, for example, Grubbs, R. H.supra; Bai et al., Org. Biomol. Chem. 3:4139-42 (2005), and Gibson etal., Chem. Comm., 1107-08 (1997). When used in reference to anesterification reaction, including a diesterification reaction, the termis intended to refer to a substance that increases the rate of thecondensation reaction without being consumed. Specific examples ofesterification catalysts for Fisher esterification are exemplifiedabove. These catalysts as well as others well known in the art for avariety of different types of esterification reactions also aredescribed in, for example, March, supra; Morrison and Boyd, supra, andCarey, F. A. and Sundberg, R. J., supra.

As used herein, the term “sufficient amount” or a grammaticallyequivalent form thereof, when used in reference to a chemical reagent ina reaction or in reference to a culture constituent is intended to meana quantity of the referenced regent or constituent that can meet thedemands of the chemical reaction or cultured microbial organism. Forexample, a sufficient amount of a catalyst refers to a quantity ofcatalyst that is adequate to increase the referenced chemical reactionrate. A sufficient amount of, for example, a carbon source in a culturemedium refers to a quantity that is adequate to support growth of acultured microbial organism.

As used herein, the term “non-naturally” when used in reference to amicrobial organism or microorganism of the invention is intended to meanthat the microbial organism has at least one genetic alteration notnormally found in a naturally occurring strain of the referencedspecies, including wild-type strains of the referenced species.“Wild-type,” or grammatical equivalents thereof, refers to the commongenotype or phenotype, or genotypes or phenotypes, of an organism as itis found in nature or in a standard laboratory stock for a givenorganism. Genetic alterations include, for example, a gene deletion orsome other functional disruption of the genetic material. Geneticalterations also include modifications introducing expressible nucleicacids encoding metabolic polypeptides, other nucleic acid additions,nucleic acid deletions and/or other functional disruption of themicrobial genetic material. Such modification include, for example,coding regions and functional fragments thereof, for heterologous,homologous or both heterologous and homologous polypeptides for thereferenced species. Exemplary metabolic polypeptides include enzymeswithin a metabolic pathway or uptake pathway for one or more carbonsources used by a referenced microbial organism such as enzymes withinthe glycolysis or the pentose phosphate pathways.

As used herein, the terms “microbial organism,” “microbe,” “microbial”or “microorganism” is intended to mean any organism that exists as amicroscopic cell that is included within the domains of archaea,bacteria or eukarya. Therefore, the term is intended to encompassprokaryotic or eukaryotic cells or organisms having a microscopic sizeand includes bacteria, archaea and eubacteria of all species as well aseukaryotic microorganisms such as yeast and fungi. The term alsoincludes cell cultures of any species that can be cultured for theproduction of a biochemical.

An isolated microbial organism refers to an organism that issubstantially free of at least one component of the referenced microbialorganism as it is found in nature. The term includes a microbialorganism that is removed from some or all components as it is found inits natural environment. The term also includes a microbial organismthat is removed from some or all components as the microbial organism isfound in non-naturally occurring environments. Therefore, an isolatedmicrobial organism is partly or completely separated from othersubstances as it is found in nature or as it is grown, stored orsubsists in non-naturally occurring environments. Specific examples ofisolated microbial organisms include partially pure microbial organism,substantially pure microbial organisms and microbial organisms culturedin a medium that is non-naturally occurring.

As used herein, the term “growth-coupled” when used in reference to thebiosynthesis of a chemical compound or biochemical is intended to meanthat the biosynthesis of the referenced molecule is an obligatoryproduct produced during the growth phase of a microbial organism.

As used herein, the term “metabolic modification” is intended to referto a biochemical reaction or transport process that is altered from itsnaturally occurring state. Metabolic modifications can include, forexample, elimination of a biochemical reaction activity by functionaldisruptions of one or more genes encoding an enzyme participating in thereaction. Sets of exemplary metabolic modifications for microbialorganisms having growth coupled production of fumaric acid areillustrated in Table 1 (starting at page 50). Individual reactionsspecified by such metabolic modifications and their corresponding genecomplements are exemplified in Table 2 (starting at page 56) for E. colias a representative microbial organism. Reactants and products utilizedin these reactions are exemplified in Table 3 (starting at page 57).

As used herein, the term “gene disruption” or grammatical equivalentsthereof, is intended to mean a genetic alteration that renders theencoded gene product inactive. The genetic alteration can be, forexample, deletion of the entire gene, deletion of a regulatory sequencerequired for transcription or translation, deletion of a portion of thegene with results in a truncated gene product or by any of variousmutation strategies that inactivate the encoded gene product. Oneparticularly useful method of gene disruption is complete gene deletionbecause it reduces or eliminates the occurrence of genetic reversions inthe non-naturally occurring microbial organisms of the invention.

As used herein, the term “stable” when used in reference togrowth-coupled production of a biochemical product is intended to referto microbial organism that can be cultured for greater than fivegenerations without loss of the coupling between growth and biochemicalsynthesis. Generally, stable growth-coupled biochemical production willbe greater than 10 generations, particularly stable growth-coupledbiochemical production will be greater than about 25 generations, andmore particularly, stable growth-coupled biochemical production will begreater than 50 generations, including indefinitely. Stablegrowth-coupled production of a biochemical can be achieved, for example,by deletion of a gene encoding an enzyme catalyzing each reaction withina set of metabolic modifications. The stability of growth-coupledproduction of a biochemical can be enhanced through multiple deletions,significantly reducing the likelihood of multiple compensatoryreversions occurring for each disrupted activity.

Those skilled in the art will understand that the metabolicmodifications exemplified herein are described with reference to E. coligenes and their corresponding metabolic reactions. However, given thecomplete genome sequencing of a wide variety of organisms and the highlevel of skill in the area of genomics, those skilled in the art willreadily be able to apply the teachings and guidance provided herein toessentially all other organisms. For example, the E. coli metabolicalterations exemplified herein can readily be applied to other speciesby incorporating the same or analogous gene disruptions in the otherspecies. Such disruptions can include, for example, genetic alterationsof species homologs, in general, and in particular, orthologs, paralogsor nonorthologous gene displacements.

An ortholog is a gene or genes that are related by vertical descent andare responsible for substantially the same or identical functions indifferent organisms. For example, mouse epoxide hydrolase and humanepoxide hydrolase can be considered orthologs for the biologicalfunction of hydrolysis of epoxides. Genes are related by verticaldescent when, for example, they share sequence similarity of sufficientamount to indicate they are homologous, or related by evolution from acommon ancestor. Genes can also be considered orthologs if they sharethree-dimensional structure but not necessarily sequence similarity, ofa sufficient amount to indicate that they have evolved from a commonancestor to the extent that the primary sequence similarity is notidentifiable. Genes that are orthologous can encode proteins withsequence similarity of about 25% to 100% amino acid sequence identity.Genes encoding proteins sharing an amino acid similarity less that 25%can also be considered to have arisen by vertical descent if theirthree-dimensional structure also shows similarities. Members of theserine protease family of enzymes, including tissue plasminogenactivator and elastase, are considered to have arisen by verticaldescent from a common ancestor.

Orthologs include genes or their encoded gene products that through, forexample, evolution, have diverged in structure or overall activity. Forexample, where one species encodes a gene product exhibiting twofunctions and where such functions have been separated into distinctgenes in a second species, the three genes and their correspondingproducts are considered to be orthologs. For the growth-coupledproduction of a biochemical product, those skilled in the art willunderstand that the orthologous gene harboring the metabolic activity tobe disrupted is to be chosen for construction of the non-naturallyoccurring microbial organism. An example of orthologs exhibitingseparable activities is where distinct activities have been separatedinto distinct gene products between two or more species or within asingle species. A specific example is the separation of elastaseproteolysis and plasminogen proteolysis, two types of serine proteaseactivity, into distinct molecules as plasminogen activator and elastase.A second example is the separation of mycoplasma 5′-3′ exonuclease andDrosophila DNA polymerase III activity. The DNA polymerase from thefirst species can be considered an ortholog to either or both of theexonuclease or the polymerase from the second species and vice versa.

In contrast, paralogs are homologs related by, for example, duplicationfollowed by evolutionary divergence and have similar or common, but notidentical functions. Paralogs can originate or derive from, for example,the same species or from a different species. For example, microsomalepoxide hydrolase (epoxide hydrolase I) and soluble epoxide hydrolase(epoxide hydrolase II) can be considered paralogs because they representtwo distinct enzymes, co-evolved from a common ancestor, that catalyzedistinct reactions and have distinct functions in the same species.Paralogs are proteins from the same species with significant sequencesimilarity to each other suggesting that they are homologous, or relatedthrough co-evolution from a common ancestor. Groups of paralogousprotein families include HipA homologs, luciferase genes, peptidases,and others.

A nonorthologous gene displacement is a nonorthologous gene from onespecies that can substitute for a referenced gene function in adifferent species. Substitution includes, for example, being able toperform substantially the same or a similar function in the species oforigin compared to the referenced function in the different species.Although generally, a nonorthologous gene displacement will beidentifiable as structurally related to a known gene encoding thereferenced function, less structurally related but functionally similargenes and their corresponding gene products nevertheless will still fallwithin the meaning of the term as it is used herein. Functionalsimilarity requires, for example, at least some structural similarity inthe active site or binding region of a nonorthologous gene compared to agene encoding the function sought to be substituted. Therefore, anonorthologous gene includes, for example, a paralog or an unrelatedgene.

Therefore, in identifying and constructing the non-naturally occurringmicrobial organisms of the invention having growth-coupled production ofa biochemical, those skilled in the art will understand with applyingthe teaching and guidance provided herein to a particular species thatthe identification of metabolic modifications should includeidentification and disruption of orthologs. To the extent that paralogsand/or nonorthologous gene displacements are present in the referencedmicrobial organism that encode an enzyme catalyzing a similar orsubstantially similar metabolic reaction, those skilled in the art alsocan eliminate these evolutionally related genes to ensure that anyfunctional redundancy in enzymatic activities do not short circuit thedesigned metabolic modifications.

Orthologs, paralogs and nonorthologous gene displacements can bedetermined by methods well known to those skilled in the art. Forexample, inspection of nucleic acid or amino acid sequences for twopolypeptides will reveal sequence identity and similarities between thecompared sequences. Based on such similarities, one skilled in the artcan determine if the similarity is sufficiently high to indicate theproteins are related through evolution from a common ancestor.Algorithms well known to those skilled in the art, such as Align, BLAST,Clustal W and others compared and determine a raw sequence similarity oridentity, and also determine the presence or significance of gaps in thesequence which can be assigned a weight or score. Such algorithms alsoare known in the art and are similarly applicable for determiningnucleotide sequence similarity or identity. Parameters for sufficientsimilarly to determine relatedness are computed based on well knownmethods for calculating statistical similarity, or the chance of findinga similar match in a random polypeptide, and the significance of thematch determined. A computer comparison of two or more sequences can, ifdesired, also be optimized visually by those skilled in the art. Relatedgene products or proteins can be expected to have a high similarity, forexample, 25% to 100% sequence identity. Proteins that are unrelated canhave an identity which is essentially the same as would be expected tooccur by chance, if a database of sufficient size is scanned (about 5%).Sequences between 5% and 24% may or may not represent sufficienthomology to conclude that the compared sequences are related. Additionalstatistical analysis to determine the significance of such matches giventhe size of the data set can be carried out to determine the relevanceof these sequences.

Exemplary parameters for determining relatedness of two or moresequences using the BLAST algorithm, for example, can be as set forthbelow. Briefly, amino acid sequence alignments can be performed usingBLASTP version 2.0.8 (Jan. 5, 1999) and the following parameters:Matrix: 0 BLOSUM62; gap open: 11; gap extension: 1; x_dropoff: 50;expect: 10.0; wordsize: 3; filter: on. Nucleic acid sequence alignmentscan be performed using BLASTN version 2.0.6 (Sep. 16, 1998) and thefollowing parameters: Match: 1; mismatch: −2; gap open: 5; gapextension: 2; x_dropoff: 50; expect: 10.0; wordsize: 11; filter: off.Those skilled in the art will know what modifications can be made to theabove parameters to either increase or decrease the stringency of thecomparison, for example, and determine the relatedness of two or moresequences.

As used herein, the term “feedstock” refers to a substance used as a rawmaterial in an industrial process. When used in reference to a cultureof microbial organisms such as a fermentation process with cells, theterm refers to the raw material used to supply a carbon or other energysource for the cells. A “renewable” feedstock refers to a renewableenergy source such as material derived from living organisms or theirmetabolic byproducts including material derived from biomass, oftenconsisting of underutilized components like chaff or stover.Agricultural products specifically grown for use as renewable feedstocksinclude, for example, corn, soybeans, switchgrass and trees such aspoplar, primarily in the United States; wheat, flaxseed and rapeseed,primarily in Europe; sugar cane in Brazil and palm oil in South-EastAsia. Therefore, the term includes the array of carbohydrates, fats andproteins derived from agricultural or animal products across the planet.

As used herein, the term “biomass” is intended to mean any plant-derivedorganic matter. Biomass available for energy on a sustainable basisincludes herbaceous and woody energy crops, agricultural food and feedcrops, agricultural crop wastes and residues, wood wastes and residues,aquatic plants, and other waste materials including some municipalwastes. Biomass feedstock compositions, uses, analytical procedures andtheoretical yields are readily available from the U.S. Department ofEnergy and can be found described, for example, at the URL1.eere.energy.gov/biomass/information_resources.html, which includes adatabase describing more than 150 exemplary kinds of biomass sources.Exemplary types of biomasses that can be used as feedstocks in themethods of the invention include cellulosic biomass, hemicellulosicbiomass and lignin feedstocks or portions of feedstocks. Such biomassfeedstocks contain, for example, carbohydrate substrates useful ascarbon sources such as glucose, sucrose, xylose, arabinose, galactose,mannose, fructose and starch. The term “biomass” also can be used torefer to a microbial population, e.g., the total microbial population ofa fermeter during and after a fermentation process.

The invention provides a method of producing acrylic acid. The methodincludes contacting fumaric acid with a sufficient amount of ethylene inthe presence of a cross-metathesis transformation catalyst to produceabout two moles of acrylic acid per mole of fumaric acid.

Olefin metathesis and cross-metathesis has been one endeavor of chemicalsynthesis research for carbon-carbon bond and intermolecularcarbon-carbon double bond formation of olefins (Grubbs, supra; Bai etal., supra, and Gibson et al., supra). However, these efforts haveresulted with varying success. The chemical structures, substituents,stereochemistry and pKa's of the reactants have led to differing results(see, for example, see Chatterjee et al. J. Am. Chem. Soc. 125:11360-70(2003)). Predictability has only been obtained after exhaustive pathwaysof experimentation even for closely related molecules.

Fumaric acid and acrylic acid are classified as olefins due to theirunsaturated hydrocarbon structure having the general formulaC_(n)H_(2n). The chemical synthesis of acrylic acid or ester formsthereof have not been achieved through olefin cross-metathesis. Incontrast however, it has been reported that dimethyl maleate, a cisisomer of dimethyl fumarate, displays low reactivity in olefinmetathesis with ethylene. (Fomine, S. and Tlenkopatchev, M. A., J. Org.Chem. 691:5189-96 (2006)). Moreover, dimethyl fumarate has been reportedto be unreactive toward terminal alkenes in cross-metathesis.(Chatterjee et al., supra). Without being bound by theory, it isgenerally understood that olefin metathesis is a substantiallyreversible process and therefore the products of a particularcross-metathesis often reflect statistical distributions governed by therelative thermodynamic energies of the various products and startingmaterials. In this respect, one skilled in the art will recognize anadditional challenge in converting fumaric acid (or its diester) toacrylic acid (or its ester). Indeed the dimerization of acrylates tofumarates is well documented.

The reactant for the acrylic acid synthesis of the invention is fumaricacid, a dicarboxylic acid having pKa's of approximately 3.0 and 4.5.Cross-metathesis of diacids is not known to have been reported. Hence,the cross-metathesis transformation of the invention of fumaric acid toacrylic acid is unanticipated based on the historic course of researchresults in olefin cross-metathesis. The use of ethylene rather thanterminal alkenes can positively affect the cross-metathesis of alkenesubstrates since the chain-carrying catalyst, [Ru═CH2], will contain theleast sterically encumbered methylidene ligand attached to the rutheniumcatalyst, thus providing for highly active catalysis (see, for example,Lloyd-Jones et al., Chemie, Int. Ed., 44, 7442-7 (2005)).

The acrylic acid synthesis method of the invention utilizescross-metathesis between fumaric acid and ethylene. Fumaric acid is adicarboxylic acid having a double bond between carbons C-2 and C-3.Cross-metathesis with ethylene splits this dicarboxylic acid into twomolecules with the net formation of a double bond in each new moleculeof acrylic acid. The net result is formation of two moles of acrylicacid per mole of fumaric acid reactant as shown in FIG. 1. Althoughfumaric acid cross-metathesis can be performed with a variety of olefinsother than ethylene, the inclusion of ethylene creates a carbon-carbondouble bond with formation of two moles of acrylic acid per mole offumaric acid. Since both reactants are symmetric, only a single product(acrylic acid) is formed.

An exemplary reaction illustrating the cross-metathesis transformationof fumaric acid to two moles of acrylic acid is shown below. Briefly,acrylic acid having the following formula

can be manufactured by reacting fumaric acid having the followingformula

with ethylene in the presence of an olefin metathesis catalyst to givethe acrylic acid of formula I.

Cross-metathesis between fumaric acid and ethylene can be performedusing a variety of synthesis methods and catalysts known in the art.Exemplary procedures and catalysts include, for example, any of thosedescribed in, for example, Grubbs, supra; Bai et al., supra; Gibson etal., supra, and Dias et al., J. Am. Chem. Soc., 119:3887-3897 (1997).Such procedures can include reaction temperatures ranging from, forexample, 0-100° C., pH ranges from about 2-10 and a variety of solventsincluding, for example, dichloromethane, dichloroethane, alcohols,water, other aqueous solutions, alcohol/water mixtures and the like.

Particularly useful catalysts include a variety of different specieswithin the ruthenium class metathesis catalysts. Exemplary rutheniumbased catalysts include, for example, phosphine-free ruthenium carbinecomplexes such as molybdenum alkoxyimidoalkylidene, rutheniumbenzylidenes and ether-tethered ruthenium alkylidene derivatives; stable16e ruthenium carbene complexes having the activebis(triphenylphosphine)-dichlororuthenium alkylidene complex, diazocompounds, ruthenium benzylidene complexes, ruthenium trichloridesprepared from late metal salts. A specific example of a phosphine-freecarbene ruthenium catalysts is[1,3-bis(2,6-dimethylphenyl)4,5-dihydroimidazol-2-ylidene]C₅H₅N)₂(Cl)₂Ru═CHPh,which is a bispyridine complex. A further specific example of aruthenium based catalyst is Cl₂(PCy₃)₂Ru═CHPh.

Other examples of cross-metathesis catalysts applicable for use in thesynthesis methods of the invention include, for example, high oxidationstate late metal complexes such as those described by Tebbe et al., J.Am. Chem. Soc., 101:5075 (1979) Wengrovius et al., J. Am. Chem. Soc.,102:4515 (1980), and Osborn et al., Chem. Commun., 431-432 (1980);titanium methylene complex or Tebbe Reagent (Pine et al., J. Am. Chem.Soc., 102:3270 (1980); unsymmetrical Tebbe complexes (Howard et al., J.Am. Chem. Soc., 102:6876 (1980); metallacyclobutane (Kress et al., J.Am. Chem. Soc., 109:899 (1987); Wengrovius et al., J. Am. Chem. Soc.,102:4515-4516 (1980), and Quignard et al., Angew. Chem. Int. Ed. Engl.,31(5):628-631 (1992)); and/or tungsten and molybdenum alkylidenecomplexes that contained bulky imido ligands (Schrock et al., J. Am.Chem. Soc., 110:1423-1435 (1988)).

Additional catalysts useful in the olefin cross-metathesis reaction ofthe invention can be exemplified, but not limited to, the following:

Selection of optimal catalysts to use in the cross-metathesis reactionsof the invention can readily be performed by those skilled in the art.For example, catalysts having a desirable activity in a particularsolution, pH and/or temperature can be selected by contacting a fumaricacid, fumarate monoester or fumarate diester substrate in the presenceof ethylene and measuring the rate of acrylic acid or acrylate esterproduct formation. Any of the catalysts exemplified above can bescreened for optimal activity as well as others known in the art.Selection of one or more optimal catalysts can be beneficial foridentifying cross-metathesis catalysts exhibiting enhanced catalyticrates.

The cross-metathesis synthesis method of the invention also can beemployed with fumarate ester or a fumarate diester and ethylene toproduce acrylate esters. In the former reaction, cross-metathesis with afumarate monoester will produce one mole of acrylic acid and one mole ofacrylate ester per mole of fumarate monoester. In the later reaction,the net result is formation of two moles of acrylate ester per mole offumarate diester reactant as shown in FIG. 2.

An exemplary reaction illustrating the cross-metathesis transformationof fumarate ester to two moles of acrylate ester is shown below.Briefly, acrylate ester having the following formula

wherein R represents straight or branched alkyl having 1 to 10 carbonatoms wherein said alkyl may be optionally and independently substitutedwith alkyl having 1 to 10 carbon atoms; phenyl; phenylalkyl; amino;hydroxy; alkylamino having 1 to 10 carbon atoms; and alkoxy having 1 to10 carbon atoms or R represents cycloalkyl having 3 to 6 ring carbonatoms wherein said ring carbon atoms may be optionally and independentlysubstituted with alkyl having 1 to 6 carbon atoms and hydroxy can bemanufactured by reacting a fumarate diester having the following formula

wherein R is defined as above with ethylene in the presence of aolefinic metathesis catalyst to give the acrylate ester of formula III.

Given the teachings and guidance provided herein, those skilled in theart will understand that the fumaric acid, fumarate diester, acrylicacid and acrylate ester of the present invention can be furthersubstituted by aliphatic and/or aromatic moieties. For example, C-2 andC-3 carbons of fumarate diester can be substituted with methyl. In thisspecific embodiment, cross-metathesis with ethylene will producemethacrylate ester. In like fashon, the C-2 and/or C-3 also can besubstituted with, for example, other alkyl such as ethyl, propyl orbutyl and subjected to cross-metathesis to yield the corresponding alkylsubstituted acrylate ester. Those skilled in the art will understandthat corresponding metathesis transformations also can be performed withfumaric acid similarly substituted. Those skilled in the art also willunderstand that the above described aliphatic and/or aromaticsubstituted moieties themselves can additionally be further substituted.

In addition to the further substitution of fumaric acid, fumaratediester, acrylic acid and acrylate ester of the present inventiondescribed above, those skilled in the art also will understand that theethylene metathesis reactant also can be further substituted. In thisregard, a wide variety of disubstitued alkeynes can be employed in thecross-metathesis reactions of the invention to yield chemical compoundsother than acrylic acid or acrylate ester. Such disubstituted alkeynescan be represented by the chemical formula RCH═CHR′, where R and R′ canbe the same or different chemical moiety, including hydrogen and anystraight or branched alkyl. A specific example of such a disubstituedalkeyne is 2-butene. By exemplification to cross-metathesis with fumaricacid, where R is hydrogen the product is acrylic acid. In comparison,where R is methyl, the product is crotanoic acid.

Fumarate mono- and diesters can be produced by a variety ofesterification methods well known in the art. A useful esterificationmethod is treatment of fumaric acid with an alcohol in the presence of amineral acid such as sulfuric acid or dry hydrogen chloride. While thechoice of alcohol will be determined by the type of ester or diesterdesired, it is to be understood that primary, secondary or tertiaryaliphatic or aromatic, substituted or unsubstituted alcohols arecontemplated by this invention. Those skilled in the art will know, orcan readily determine, what alcohol or alcohols can be selected for usewith a particular type of ester or diester.

A particularly useful esterification method is treatment of fumaric acidhaving the following formula

with an alcohol having the formula ROH, wherein R is represented bystraight or branched alkyl having 1 to 10 carbon atoms wherein saidalkyl may be optionally and independently substituted with alkyl having1 to 10 carbon atoms; phenyl; phenylalkyl; amino; hydroxy; alkylaminohaving 1 to 10 carbon atoms; and alkoxy having 1 to 10 carbon atoms or Rrepresents cycloalkyl having 3 to 6 ring carbon atoms wherein said ringcarbon atoms may be optionally and independently substituted with alkylhaving 1 to 6 carbon atoms and hydroxy in the presence of a mineralacid.

While the above esterification method describes treatment of fumaricacid with an alcohol in the presence of a mineral acid to arrive at thefumaric diester, it is also understood that the fumaric acid can beconverted into an acid chloride which can then be treated with analcohol to arrive at the ester or diester. One benefit of the two-stepreaction as opposed to the direct esterification method is that thereversibility of the direct ester route is avoided.

The invention also provides a process for producing acrylic acid. Theprocess includes: (a) culturing in a sufficient amount of nutrients andmedia a non-naturally occurring microbial organism having a set ofmetabolic modifications obligatorily coupling fumaric acid production togrowth of the microbial organism, the set of metabolic modificationsincludes disruption of at least one of the gene sets having: (1) fumABC,zwf, purU, or (2) fumABC, zwf, glyA, or an ortholog thereof, to producestable growth-coupled production of fumaric acid, and (b) contacting thefumaric acid with a sufficient amount of ethylene in the presence of across-metathesis transformation catalyst to produce about two moles ofacrylic acid per mole of fumaric acid.

A further embodiment of the invention includes coupling fumaric acidsubstrate biosynthesis with chemical synthesis of acrylic acid oracrylate esters in an integrated process. FIG. 3 illustrates oneapproach for integrated production of acrylic acid from the biosynthesisof fumaric acid substrate. Those skilled in the art will understand thatalthough the integration of substrate production through a bioprocesssuch as fermentation and final product manufacture through one or morechemical synthesis procedures is illustrated herein with respect tobiosynthesis of fumaric acid, given the teachings and guidance providedherein, any combination or permutation of biosynthesis to one or moreintermediates and chemical synthesis of final product can beaccomplished using the process of the invention. Given the teachings andguidance provided herein, those skilled in the art also will understandthat a chemical synthesis step can be utilized in synthesis of one ormore intermediates to a final product. Similarly, those skilled in theart also can employ a genetic modifications and biosynthesis toaccomplish the conversion of fumaric acid to acrylic acid. Accordingly,the integrated process shown in FIG. 3 illustrating biosynthesis from aglucose carbon source to the acrylic acid using fumaric acid as anintermediate substrate is exemplary. Therefore, genetic modificationsresulting in entry and flux through any portion of glycolysis, TCA orother metabolic pathways that result in increased fumaric acidproduction can be employed in a process of the invention for productionof acrylic acid and acrylate esters of the invention.

Useful embodiments of an integrated process of the invention is thebioproduction of a genetically engineered product which is a substrateor intermediate to olefin metathesis. In this regard, fermentation ofnon-naturally occurring organisms modified to biosynthesize specificproducts are particularly useful sources for chemical compounds such asfumaric acid and other olefins. Given the teachings and guidanceprovided herein, those skilled in the art will understand that theintegrated process exemplified herein with respect to the olefin fumaricacid and the cross-metathesis transformation to acrylic acid can beequally applied to produce essentially any olefin of interest. Sucholefins can be coupled to a metathesis transformation for the chemicalsynthesis of a wide variety of other olefins. Those skilled in the artalso will understand that the integrated process coupling bioproductionby, for example, fermentation of an olefin substrate to a metathesistransformation also can be employed in the production of an olefinintermediate. The intermediate can be chemically converted to an olefinthat can serve as a substrate for olefin metathesis.

Specific examples of coupling an olefin product of bioproduction suchfermentation to olefin metathesis is the production of the olefinfumaric acid and cross-metathesis to acrylic acid and other compounds asdescribed previously. Specific examples of coupling a product ofbioproduction such as fermentation to yield an intermediate to olefinmetathesis are exemplified below. Chemical conversion of suchintermediates to an olefin yields substrates useful in olefin metathesisand also can be performed in an integrated process as describedpreviously and below. For example, 3-hydroxypropionic acid (3-HP) can beproduced by fermentation of 3-HP producing microbal organisms anddehydrated to the olefin acrylic acid. The acrylic acid can be subjectedto metathesis with an olefin of interest to produce a desired olifenproduct. Similarly, 2,3-butane diol also can be produced by fermentationusing the teachings and guidance provided herein. The 2,3-butane diolintermediate can be further dehydrated into butadiene which can beemployed as an olefin substrate to make a wide range of olefin productsthrough metathesis transformation.

Therefore, the invention provides a process for producing an olefin. Theprocess includes: (a) culturing by fermentation in a sufficient amountof nutrients and media a microbal organism that produces a first olefin,and (b) contacting the first olefin with a sufficient amount of adisubstitued alkeyne in the presence of an olefin metathesistransformation catalyst to produce second, different olefin. Thedisubstituted alkeyne can be ethylene. The microbial organism of can be,for example, a non-naturally occurring microbal organism such as anorganism genetically engineered to produce the first olefin, or anaturally occurring microbial organism such as an organism thatnaturally produces the first olefin.

The invention further provides a process for producing an olefin. Theprocess includes: (a) culturing by fermentation in a sufficient amountof nutrients and media a microbal organism that produces an olefinintermediate; (b) performing a chemical modification to convert theolefin intermediate to a first olefin, and (c) contacting the firstolefin with a sufficient amount of a disubstitued alkeyne in thepresence of an olefin metathesis transformation catalyst to producesecond, different olefin. The chemical modification can be, for example,dehydrogenation. The disubstituted alkeyne can be ethylene. Themicrobial organism of can be, for example, a non-naturally occurringmicrobial organism such as an organism genetically engineered to producethe olefin intermediate, or a naturally occurring microbial organismsuch as an organism that naturally produces the olefin intermediate.

Step 1 illustrated in FIG. 3 exemplifies biological production offumaric acid, which derives from the TCA cycle and is a commonintermediate of central cellular metabolism. Central metabolites areparticularly useful targets for metabolic engineering as they are oftenconstitutively produced during basal metabolism.

Step 2 of the integrated process illustrated in FIG. 3 exemplifies thecoupling of olefin cross-metathesis involving ethylene as describedpreviously and shown in FIG. 1. In one embodiment, coupling of thebioproduction of fumaric acid and cross-metathesis is performed bydirect addition of a selected cross-metathesis catalyst and ethylene tothe fumaric acid culture or fermentation broth. Such direct coupling isan efficient and streamlined manufacturing process of acrylic acid.Olefin metathesis based upon ruthenium catalysts has been shown toperform well in water (see, for example, Lynn et al., cJ. Am. Chem.Soc., 118:784-90 (1996) and Lynn et al. J. Am. Chem. Soc. 120:1627-28(1998). In another embodiment, fumaric acid can be isolated from theculture medium or fermentation broth and reacted separately with across-metathesis catalyst and ethylene to synthesize acrylic acid.

Integrating biosynthesis of fumaric acid and chemical cross-metathesistransformation with ethylene, for example, to produce acrylic acid isadditionally useful because it results in a highly efficient conversionof substrate carbon (e.g., glucose or sucrose) into the desired product(e.g., acrylic acid). Similarly, coupling of esterification, includingdiesterification, of fumaric acid to fumarate mono or diester alsoyields the same carbon utilization efficiencies. For example, carbonfrom 1 mole of glucose entering the glycolysis metabolic pathwayprovides two moles of phosphoenol pyruvate (PEP), which reacts withcarbon dioxide via PEP carboxylase to result in a maximum theoreticalyield of approximately 2.0 moles of fumaric acid. Upon cross-metathesiswith ethylene, each mole of fumaric acid yields two moles of acrylicacid, resulting a process where one mole of glucose and 2.0 moles ofethylene are converted into up to four moles of acrylic acid.

Another particularly useful attribute of the integrated process of theinvention illustrated in FIG. 3 is that any thermodynamic constraintsencountered in the production of acrylic acid directly from glucose byfermentation as well as possible toxicity of acrylic acid to the hostorganism can be avoided. The integrated process of the inventionbiologically produces fumaric acid, which is a normal metabolicintermediate, and then transforms fumaric acid to acrylic acid in apost-culture or post-fermentation step, thus avoiding exposure of theproduction organisms to, for example, a lethal fermentation product.

In one embodiment, the fumaric acid producing microbial organisms thatcan be used in an integrated process of the invention include isolatedorganisms that naturally produce fumaric acid. In another embodiment,the fumaric acid producing microbial cells can be genetically engineeredfor enhanced expression of fumaric acid. Particularly useful engineeredmicrobial organisms include metabolic modifications that couple organismgrowth to product biosynthesis. For the integrated production process ofacrylic acid and/or acrylate ester of the invention, the biosyntheticproduct is fumaric acid.

Growth coupled production of fumaric acid can be accomplished by, forexample, identifying metabolic modifications that obligatory couplefumaric acid to growth. Particularly useful methods that can be employedto accurately predict biological behavior in response to genetic changesinclude in silico methods such as those exemplified further below anddescribed in, for example, U.S. patent publications US 2002/0012939, US2003/0224363, US 2004/0029149, US 2004/0072723, US 2003/0059792, US2002/0168654 and US 2004/0009466, and in U.S. Pat. No. 7,127,379. Suchmethod include in silico construction, optimization and modifications ofmetabolic and regulatory networks including, for example, identificationof gene sets that when disrupted obligatory couple growth to fumaricacid production. Once identified, the set of reactions that are to bedisrupted in order to achieve growth-coupled fumaric acid production areimplemented in the target cell or organism by functional disruption ofat least one gene encoding each metabolic reaction within the set.

As described previously, one particularly useful means to achievefunctional disruption of the reaction set is by deletion of eachencoding gene. However, in some instances, it can be beneficial todisrupt the reaction by other genetic aberrations including, forexample, mutation, deletion of regulatory regions such as promoters orcis binding sites for regulatory factors, or by truncation of the codingsequence at any of a number of locations. These latter aberrations,resulting in less than total deletion of the gene set can be useful, forexample, when rapid assessments of the fumaric acid coupling are desiredor when genetic reversion is less likely to occur. Those skilled in theart also will understand that any molecular design and recombinantimplementation, for example, can be used to add, delete or substituteone or more genes encoding enzymes in a metabolic pathway to confer adesired activity onto the host organism. Therefore, although thenon-naturally occurring microbial organisms of the invention areexemplified herein with respect to disruption of genes to generate ametabolic network obligatory coupling fumaric acid to growth, thoseskilled in the art will understand that the non-naturally occurringmicrobial organisms of the invention also include genetic modificationsthat confer a desired metabolic activity by, for example, introductionof one or more metabolic activities into a host microbial organism.

Briefly, with respect to introducing one or more desired metabolicactivities, those skilled in the art will understand that the number ofencoding nucleic acids to introduce in an expressible form will parallelthe deficiencies in the target pathway to be constructed. Therefore, oneor more host microbial organisms for use in the integrated process ofthe invention can have one, two, three, four, five or six encodingnucleic acids encoding the enzymes constituting the target productbiosynthetic pathway or pathways. In some embodiments, the hostmicrobial organism or organisms also can include other geneticmodifications that facilitate or optimize target product biosynthesis orthat confer other useful functions onto the host microbial organism.

Sources of encoding nucleic acids which can be used for generating thevarious metabolic modifications including, for example, expression ofheterologous metabolic polypeptides, effecting targeted disruptions ofmetabolic genes or for other recombinantly engineered modificationsexemplified herein can include, for example, any species where theencoded gene product is capable of catalyzing the referenced reaction oractivity. Such species include both prokaryotic and eukaryotic organismsincluding, but not limited to, bacteria, archaea, eubacteria, animal,mammal, including human.

Methods for constructing and testing the expression levels of any of thenon-naturally occurring microbial organisms, including those modified tosynthesize an encoding polypeptide as well as conformation thatdisrupted genes reduce or eliminate expression of the encodedpolypeptide, can be performed, for example, by recombinant proceduresand detection methods well known in the art. Such methods can be founddescribed in, for example, Sambrook et al., Molecular Cloning: ALaboratory Manual, Third Ed., Cold Spring Harbor Laboratory, New York(2001); Ausubel et al., Current Protocols in Molecular Biology, JohnWiley and Sons, Baltimore, Md. (1999).

Employing the methods exemplified above and further below, metabolicmodifications have been identified that obligatory couple the productionof fumaric acid to microbial organism growth. Microbial organism strainsconstructed with the identified metabolic modifications produce elevatedlevels of fumaric acid during the exponential growth phase. Thesestrains can be beneficially used for the commercial production offumaric acid in, for example, continuous fermentation process withoutbeing subjected to the negative selective pressures describedpreviously. Such production can be coupled with cross-metathesistransformation or with diesterification followed by cross-metathesistransformation in an integrated process for efficient production ofacrylic acid and acrylate esters, respectfully.

Non-naturally occurring microbial organisms of the invention includebacteria, yeast, fungus or any of a variety of other microbial organismsapplicable to fermentation processes. Exemplary bacteria include speciesselected from E. coli, A. succiniciproducens, A. succinogenes, M.succiniciproducens, R. etli, Bacillus subtilis, Corynebacteriumglutamicum, Gluconobacter oxydans, Zymomonas mobilis, Lactococcuslactis, Lactobacillus plantarum, Strep tomyces coelicolor, Clostridiumacetobutylicum, Pseudomonas fluorescens, and Pseudomonas putida.Exemplary yeasts include species selected from Saccharomyces cerevisiae,Schizosaccharomyces pombe, Kluyveromyces lactis, Kluyveromycesmarxianus, Aspergillus terreus, Aspergillus niger, Rhizopus arrhizus,Rhizopus oryzae, and Pichia pastoris. With respect to the integratedprocess of the invention described further below, microbial organismsthat tolerate low pH are particularly useful due to the avoidance of anydesired neutralization steps and the lowering of salt formationassociated with acid production using acid-intolerant organisms.Microbial organisms tolerant to pH of about 3.0 or less can be used ifthese characteristics are desirable in an integrated process ofproducing acrylic acid and/or acrylate esters. However, microbialorganisms that tolerate pH values of about 6.0, 5.5, 5.0. 4.5, 4.0 or3.5 or less, including all pH values in between or below these exemplaryvalues, also can be used as well.

The microbial organisms having growth-coupled fumaric acid productionare exemplified herein with reference to an E. coli genetic background.However, with the complete genome sequence available for now more than550 species (with more than half of these available on public databasessuch as the NCBI), including 395 microbial organism genomes and avariety of yeast, fungi, plant, and mammalian genomes, theidentification of an alternate species homolog for one or more genes,including for example, orthologs, paralogs and nonorthologous genedisplacements, and the interchange of genetic alterations betweenorganisms is routine and well known in the art. Accordingly, themetabolic modifications enabling growth-coupled production of fumaricacid described herein with reference to a particular organism such as E.coli can be readily applied to other microbial organisms, includingprokaryotic and eukaryotic organisms alike. Given the teachings andguidance provided herein, those skilled in the art will know that ametabolic modification exemplified in one organism can be appliedequally to other organisms.

For example, fumaric acid production can be coupled to exponentialgrowth in E. coli by deletion or functional removal of one or more genesencoding enzymes catalyzing the reaction referred to herein as FUM, oneor more genes encoding enzymes catalyzing the reaction referred toherein as PGDH, and one or more genes encoding enzymes catalyzing thereaction referred to herein as FTHFD. As shown in Table 2, E. coli genesthat encode an enzyme catalyzing the FUM reaction is fumABC or b1611,b1612 and b4122. Also, shown in Table 2 is an E. coli gene that encodesan enzyme catalyzing the PGDH reaction. This PDGH associated gene is gndor b2029. Similarly, the E. coli gene encoding the enzyme catalyzing theFTHFD reaction is purU or b1232. To produce a metabolically engineeredE. coli exhibiting growth coupled succinate production, genes encodingat least one enzyme catalyzing each of the FUM, PGDH and FTHFD reactionshave to be functionally disrupted. The disruption of these genes shouldinclude orthologs. Such a disruption can occur, for example, by deletingany of the fumAB or C genes (b1611, b1612 and b4122) and the gnd (b2029)and the purU (b1232) genes. For the growth-coupled production of fumaricacid in a cell or organism other then E. coli the genes encodingcomparable reactions for FUM, PGDH and FTHFD in the species of interestcan be functionally disrupted. For those organisms having analogousmetabolic pathways such disruption can be accomplished by deleting, forexample, the species homologue to the fumAB or C genes (b1611, b1612 andb4122) and the gnd (b2029) and the puru (b1232) genes.

As described previously, such homologues can include othologs and/ornonorthologous gene displacements. In some instances, such as when asubstitute metabolic pathway exists in the species of interest,functional disruption can be accomplished by, for example, deletion of aparalog that catalyzes a similar, yet non-identical metabolic reactionwhich replaces the referenced reaction. Because certain differencesamong metabolic networks between different organisms, those skilled inthe art will understand that the actual genes disrupted betweendifferent organisms may differ. However, the given the teachings andguidance provided herein, those skilled in the art also will understandthat the methods of the invention can be applied to all microbialorganisms to identify the cognate metabolic modifications betweenorganisms and to construct an organism in a species of interest thatwill enhance the coupling of fumaric acid biosynthesis to growth.

The fumaric acid producing organisms of the invention will be describedherein with general reference to the metabolic reaction, reactant orproduct thereof, or with specific reference to one or more genesassociated with the referenced metabolic reaction, reactant or product.Unless otherwise expressly stated herein, those skilled in the art willunderstand that reference to a reaction also constitutes reference tothe reactants and products of the reaction. Similarly, unless otherwiseexpressly stated herein, reference to a reactant or product alsoreferences the reaction and that reference to any of these metabolicconstitutes also references the gene or genes encoding the enzymes thatcatalyze the referenced reaction, reactant or product. Likewise, giventhe well known fields of metabolic biochemistry, enzymology andgenomics, reference herein to a gene also constitutes a reference to thecorresponding encoded enzyme and the reaction it catalyzes as well asthe reactants and products of the reaction. Exemplary reactions,reaction nomenclature, reactants, products, cofactors and genes encodingenzymes catalyzing a reaction involved in the growth-coupled productionof fumaric acid are set forth in Tables 1, 2 and 3. Sets of metabolicmodifications or transformations that result in elevated levels offumaric acid biosynthesis during exponential growth are exemplified inTable 1. Each modification within a set corresponds to the requisitemetabolic reaction that should be functionally disrupted. Functionaldisruption of all reactions within each set results in the obligatoryproduction of fumaric acid by the engineered strain during the growthphase. The corresponding reactions to the referenced modifications inTable 1, and the gene or genes that potentially encode them in E. coli,are set forth in Table 2. Table 3 provides the full biochemical namesfor the reactants, cofactors and products referenced in the reactions ofTable 2.

For example, for each strain exemplified in Table 1, the metabolicmodifications that can be generated for growth coupled fumaric acidproduction are shown in each row. These modifications include thefunctional disruption of from one to six or more reactions. Inparticular, 187 strains are exemplified in Table 1 that havenon-naturally occurring metabolic genotypes. Each of these non-naturallyoccurring modifications result in an enhanced level of fumaric acidproduction during the exponential growth phase of the microbial organismcompared to a wild-type strain, under appropriate culture conditions.Appropriate conditions include, for example, those exemplified furtherbelow in the Examples such as particular carbon sources or reactantavailabilities and/or adaptive evolution.

Given the teachings and guidance provided herein, those skilled in theart will understand that to disrupt an enzymatic reaction it isnecessary to disrupt the catalytic activity of the one or more enzymesinvolved in the reaction. Disruption can occur by a variety of meansincluding, for example, deletion of an encoding gene or incorporation ofa genetic alteration in one or more of the encoding gene sequences. Theencoding genes targeted for disruption can be one, some, or all of thegenes encoding enzymes involved in the catalytic activity. For example,where a single enzyme is involved in a targeted catalytic activitydisruption can occur by a genetic alteration that reduces or destroysthe catalytic activity of the encoded gene product. Similarly, where thesingle enzyme is multimeric, including heteromeric, disruption can occurby a genetic alteration that reduces or destroys the function of one orall subunits of the encoded gene products. Destruction of activity canbe accomplished by loss of the binding activity of one or more subunitsin order to form an active complex, by destruction of the catalyticsubunit of the multimeric complex or by both. Other functions ofmultimeric protein association and activity also can be targeted inorder to disrupt a metabolic reaction of the invention. Such otherfunctions are well known to those skilled in the art. Further, some orall of the functions of a single polypeptide or multimeric complex canbe disrupted according to the invention in order to reduce or abolishthe catalytic activity of one or more enzymes involved in a reaction ormetabolic modification of the invention. Similarly, some or all ofenzymes involved in a reaction or metabolic modification of theinvention can be disrupted so long as the targeted reaction isdestroyed.

Given the teachings and guidance provided herein, those skilled in theart also will understand that an enzymatic reaction can be disrupted byreducing or eliminating reactions encoded by a common gene and/or by oneor more orthologs of that gene exhibiting similar or substantially thesame activity. Reduction of both the common gene and all orthologs canlead to complete abolishment of any catalytic activity of a targetedreaction. However, disruption of either the common gene or one or moreorthologs can lead to a reduction in the catalytic activity of thetargeted reaction sufficient to promote coupling of growth to fumaricacid biosynthesis. Exemplified herein are both the common genes encodingcatalytic activities for a variety of metabolic modifications as well astheir orthologs. Those skilled in the art will understand thatdisruption of some or all of the genes encoding a enzyme of a targetedmetabolic reaction can be practiced in the methods of the invention andincorporated into the non-naturally occurring microbial organisms of theinvention in order to achieve the growth-coupled fumaric acidproduction.

Therefore, the invention further provides a non-naturally occurringmicrobial organism having a set of metabolic modifications obligatorycoupling fumaric acid production to growth of said microbial organism.The set of metabolic modifications include disruption of one or moregenes encoding an enzyme catalyzing each reaction selected from the setof reactions including:

(a) FUM (fumABC), PGDH (gnd), FTHFD (purU); (Strain A)

(b) FUM (fumABC), PGDH (gnd), FTHFD ((purU), ACKr (ackA-pta); (Strain B)

(c) FUM (fumABC), PGDH (gnd), GHMT2 (glyA); (Strain C)

(d) FUM (fumABC), PGDH (gnd), GHMT2(glyA), GLCpts (ptsG); (Strain D)

(e) FUM (fumABC), PGDH (gnd), FTHFD (purU), GLUDy (gdhA); (Strain E)

(f) FUM (fumABC), PGDH (gnd), FTHFD (purU), THD2 (pntAB); (Strain F)

(g) FUM (fumABC), FTHFD (purU), THD2 (pntAB), ACKr (ackA-pta), PGM(yibO), PGL (ybhE); (Strain G)

wherein the microbial organism exhibits stable growth-coupled productionof fumaric acid. The common names for the genes encoding the enzymesresponsible for catalyzing the specified reactions are shown inparenthesis.

In the non-naturally occurring microbial organisms having the metabolicmodification (a) FUM, PGDH, FTHFD, (b) FUM, PGDH, FTHFD, ACKr or (d)FUM, PGDH, GHMT2, GLCPts, fumA, fumB, and fumC are genes encodingseparate enzymes potentially capable of carrying out the FUM reaction.Thus at least one and possibly all three, fumA, fumB, and fumC must beremoved to prevent FUM from uncoupling fumaric acid production from cellgrowth. Alternatively, the reaction GLCpts is carried out by a proteincomplex encoded by multiple genes. Deleting one or a combination ofgenes from the pts gene cluster, is thus sufficient for disrupting theGLCpts reaction.

Briefly, with respect to the genes exemplified above and theirrelationship to their cognate subunits within multimeric complexes,their orthologs and the reactions catalyzed by their gene products, FUMby the enzyme encoded by b1611, b1612, and b4122, PGDH is encoded by theproduct of one gene, b2029 (gnd) and FTHFD activity by purU (b1232).ACKr is encoded by the product of one gene, b2296(ackA-pta), which hasan ortholog b3115. GHMT2 is encoded by the product of the gene: b2551(glyA). GLCpts activity requires enzyme subunits encoded by nine genes:b2415, b2416, b2417, b1817, b1818, b1819, b1101, b0679, and b1621(represented collectively as ptsG). THD2 is the reaction product of acomplex encoded by the genes pntA (b1603) and pntB (b1602). Since thereactions THD2 and GLCpts are carried out by protein complexes encodedby multiple genes, deleting one or a combination of genes from the ptsand pnt gene clusters is thus sufficient for disrupting the reactions.GLUDy is catalyzed by an enzyme encoded by the gene gdhA (b1761). ThePGM and PGL activities are a function of the enzymes encoded by b3612and b0767 respectively.

As described above, functional disruption of the above metabolicreactions to yield fumaric acid producing microbial organisms also canbe accomplished by substituting the gnd gene with the zwf gene forelimination of the PGDH reaction. Employing this gene substitutionyields the following metabolic modifications which disrupt the enzymescatalyzing the reactions set forth for Strains A-G, above:

(a) fumABC, zwf, purU (strain A)

(b) fumABC, zwf, purU, ackA-pta (B)

(c) fumABC, zwf, glyA (C)

(d) fumABC, zwf, glyA, ptsG (D)

(e) fumABC, zwf, purU, gdhA (E)

(f) fumABC, zwf, purU, pntAB (F)

(g) fumABC, pntAB, purU, ackA-pta, yibO, ybhE (G)

Two common sets of gene deletions within the above exemplified strainsthat can be used for example to generate fumaric acid producingmicrobial organism include:

(a) fumABC, zwf, purU

(b) fumABC, zwf, glyA.

Accordingly, a non-naturally occurring microbial organism having a setof metabolic modifications coupling fumaric acid production to growth ofthe microbial organism is provided where the set of metabolicmodifications includes disruption of one or more genes selected from thegene sets including: (a) fumABC, zwf, purU and (b) fumABC, zwf, glyA, oran ortholog thereof, wherein the microbial organism exhibits stablegrowth-coupled production of fumaric acid. Additionally provided is anon-naturally occurring microbial organism having the genes encoding themetabolic modification (a) fumABC, zwf, purU that further includesdisruption of at least one gene selected from (1) ackA-pta, (2) gdhA,(3) pntAB and (4) ackA-pta, yibO, ythE.

Given the teachings and guidance provided herein, those skilled in theart will understand that a wide variety of combinations and permutationsexist for the non-naturally occurring microbial organisms useful in themethods and processes of the invention. One computational particularlyuseful method for identifying and designing metabolic modificationsfavoring biosynthesis of a product is the OptKnock computationalframework, Burgard et al., Biotechnol Bioeng, 84: 647-57 (2003).OptKnock is a metabolic modeling and simulation program that suggestsgene deletion strategies that result in genetically stable microbialorganisms which overproduce the target product. Specifically, theframework examines the complete metabolic and/or biochemical network ofa microbial organism in order to suggest genetic manipulations thatforce the desired biochemical to become an obligatory byproduct of cellgrowth. By coupling biochemical production with cell growth throughstrategically placed gene deletions or other functional gene disruption,the growth selection pressures imposed on the engineered strains afterlong periods of time in a bioreactor lead to improvements in performanceas a result of the compulsory growth-coupled biochemical production.Lastly, when gene deletions are constructed there is a negligiblepossibility of the designed strains reverting to their wild-type statesbecause the genes selected by OptKnock are to be completely removed fromthe genome. Therefore, this computational methodology can be used toeither identify alternative pathways that lead to biosynthesis offumaric acid or used in connection with the non-naturally occurringmicrobial organisms for further optimization of fumaric acidbiosynthesis.

Briefly, OptKnock is a term used herein to refer to a computationalmethod and system for modeling cellular metabolism. The OptKnock programrelates to a framework of models and methods that incorporate particularconstraints into flux balance analysis (FBA) models. These constraintsinclude, for example, qualitative kinetic information, qualitativeregulatory information, and/or DNA microarray experimental data.OptKnock also computes solutions to various metabolic problems by, forexample, tightening the flux boundaries derived through flux balancemodels and subsequently probing the performance limits of metabolicnetworks in the presence of gene additions or deletions. OptKnockcomputational framework allows the construction of model formulationsthat enable an effective query of the performance limits of metabolicnetworks and provides methods for solving the resulting mixed-integerlinear programming problems. The metabolic modeling and simulationmethods referred to herein as OptKnock are described in, for example,U.S. patent application Ser. No. 10/043,440, filed Jan. 10, 2002, and inInternational Patent No. PCT/US02/00660, filed Jan. 10, 2002.

Another computational method for identifying and designing metabolicmodifications favoring biosynthetic production of a product is metabolicmodeling and simulation system termed SimPheny®. This computationalmethod and system is described in, for example, U.S. patent applicationSer. No. 10/173,547, filed Jun. 14, 2002, and in International PatentApplication No. PCT/US03/18838, filed Jun. 13, 2003.

SimPheny® is a computational system that can be used to produce anetwork model in silico and to simulate the flux of mass, energy orcharge through the chemical reactions of a biological system to define asolution space that contains any and all possible functionalities of thechemical reactions in the system, thereby determining a range of allowedactivities for the biological system. This approach is referred to asconstraints-based modeling because the solution space is defined byconstraints such as the known stoichiometry of the included reactions aswell as reaction thermodynamic and capacity constraints associated withmaximum fluxes through reactions. The space defined by these constraintscan be interrogated to determine the phenotypic capabilities andbehavior of the biological system or of its biochemical components.Analysis methods such as convex analysis, linear programming and thecalculation of extreme pathways as described, for example, in Schillinget al., J. Theor. Biol. 203:229-248 (2000); Schilling et al., Biotech.Bioeng. 71:286-306 (2000) and Schilling et al., Biotech. Prog.15:288-295 (1999), can be used to determine such phenotypiccapabilities. As described in the Examples below, this computationmethodology was used to identify and analyze the feasible as well as theoptimal 4-HB biosynthetic pathways in 4-HB non-producing microbialorganisms.

As described above, one constraints-based method used in thecomputational programs applicable to the invention is flux balanceanalysis. Flux balance analysis is based on flux balancing in a steadystate condition and can be performed as described in, for example, Varmaand Palsson, Biotech. Bioeng. 12:994-998 (1994). Flux balance approacheshave been applied to reaction networks to simulate or predict systemicproperties of, for example, adipocyte metabolism as described in Felland Small, J. Biochem. 138:781-786 (1986), acetate secretion from E.coli under ATP maximization conditions as described in Majewski andDomach, Biotech. Bioeng. 35:732-738 (1990) or ethanol secretion by yeastas described in Vanrolleghem et al., Biotech. Prog. 12:434-448 (1996).Additionally, this approach can be used to predict or simulate thegrowth of E. coli on a variety of single-carbon sources as well as themetabolism of H. influenzae as described in Edwards and Palsson, Proc.Natl. Acad. Sci. 97:5528-5533 (2000), Edwards and Palsson, J. Bio. Chem.274:17410-17416 (1999) and Edwards et al., Nature Biotech. 19:125-130(2001).

Given the teachings and guidance provided herein, those skilled in theart will be able to apply various computational frameworks for metabolicmodeling and simulation to design and implement biosynthesis of fumaricacid or other desired chemical substrates in host microbial organismsother than E. coli and yeast. Such metabolic modeling and simulationmethods include, for example, the computational systems exemplifiedabove as SimPheny® and OptKnock.

The non-naturally occurring microbial organisms of the invention can beemployed in the integrated process of the invention for growth-coupledproduction of fumaric acid coupled with transformation to acrylic acidor diesterification followed by transformation to acrylate ester.Essentially any quantity of fumaric acid substrate, including commercialquantities, can be synthesized using the growth-coupled fumaric acidproducing microbial organisms of the invention. Because the microbialorganisms used in the process of the invention obligatory couple fumaricacid to growth, continuous or near-continuous growth processes areparticularly useful for biosynthetic production of fumaric acid. Suchcontinuous and/or near continuous growth processes are exemplifiedfurther below. Continuous and/or near-continuous microbial organismgrowth processes also are well known in the art. Briefly, continuousand/or near-continuous growth processes involve maintaining themicrobial organism in an exponential growth or logarythimic phase.Procedures include using apparatuses such as the Evolugatorm evolutionmachine (Evolugate LLC, Gainesville, Fla.), fermentors and the like.Additionally, shake flask fermentation and growth under microaerobicconditions also can be employed. Given the teachings and guidanceprovided herein those skilled in the art will understand that thegrowth-coupled fumaric acid producing microbial organisms can beemployed in a variety of different settings under a variety of differentconditions using a variety of different processes and/or apparatuseswell known in the art.

Generally, the continuous and/or near-continuous production of fumaricacid will include culturing a non-naturally occurring growth-coupledfumaric acid producing organism of the invention in sufficient nutrientsand medium to sustain and/or nearly sustain growth in an exponentialphase. Continuous culture under such conditions can be grown, forexample, for a day, 2, 3, 4, 5, 6 or 7 days or more. Additionally,continuous cultures can include time durations of 1 week, 2, 3, 4 or 5or more weeks and up to several months. It is to be understood that thecontinuous and/or near-continuous culture conditions also can includeall time intervals in between these exemplary periods.

One particularly useful method for large scale bioproduction of achemical product is fermentation. Briefly, fermentation procedures arewell known in the art. Fermentation of a set of complementarymetabolizing organisms in general, and for example, for the biosyntheticproduction of a target product of the invention such as a chemicalcompound can be utilized in, for example, batch fermentation, fed-batchfermentation; fed-batch fermentation or continuous fermentation. Inaddition, any of these methods of fermentation also can be coupled towell know separation methods applicable to fermentation procedures suchas batch separation or continuous separation. Exemplary combinations offermentation and separation methods applicable for bioproduction of atarget chemical compound of the invention such as fumaric acid include,for example, batch fermentation and batch separation; batch fermentationand continuous separation; fed-batch fermentation and batch separation;fed-batch fermentation and continuous separation; continuousfermentation and batch separation or continuous fermentation andcontinuous separation.

Examples of batch and continuous fermentation procedures are well knownin the art. An exemplary procedure for fed-batch fermentation and batchseparation includes culturing a production organism such as a set ofcomplementary metabolizing organisms in a 10 L bioreactor sparged withan N₂/CO₂ mixture, using 5 L broth containing 5 g/L potassium phosphate,2.5 g/L ammonium chloride, 0.5 g/L magnesium sulfate, and 30 g/L cornsteep liquor, and an initial first and second carbon sourceconcentration of 20 g/L. As the CMOs grow and utilize the carbonsources, additional 70% carbon source mixture is fed into the bioreactorat a rate approximately balancing carbon source consumption. Thetemperature of the bioreactor is generally maintained at 30° C. Growthcontinues for approximately 24 hours or more, the target chemicalcompound reaches a concentration of between 20-200 g/L, with the celldensity being between about 5 and 10 g/L. Upon completion of thecultivation period, the fermenter contents can be passed through a cellseparation unit such as a centrifuge to remove cells and cell debris,and the fermentation broth can be transferred to a product separationsunit. Isolation of the target chemical compound can take place bystandard separations procedures well known in the art to separateorganic products from dilute aqueous solutions, such as liquid-liquidextraction using a water immiscible organic solvent (e.g., toluene) toprovide an organic solution of the target chemical compound. Theresulting solution can then be subjected to standard distillationmethods to remove and recycle the organic solvent and to isolate thetarget chemical compound having a known boiling point as a purifiedliquid, for example.

An exemplary procedure for continuous fermentation and continuousseparation includes initially culturing a production organism such as aset of complementary metabolizing organisms in batch mode using, forexample, a bioreactor apparatus and medium composition exemplifiedabove, except that the initial at least first and second carbon sourceis about 30-50 g/L. When the carbon source is exhausted, feed medium ofthe same composition is supplied continuously at a rate of between about0.5 L/hr and 1 L/hr, and liquid is withdrawn at the same rate. Thetarget chemical compound concentration in the bioreactor generallyremains constant at 30-40 g/L, and the cell density generally remainsconstant at between about 3-5 g/L. Temperature is generally maintainedat 30° C., and the pH is generally maintained at about 4.5 usingconcentrated NaOH and HCl, as required. The bioreactor can be operatedcontinuously, for example, for about one month, with samples taken everyday or as needed to assure consistency of the target chemical compoundconcentration. In continuous mode, fermenter contents are constantlyremoved as new feed medium is supplied. The exit stream, containingcells, medium, and target chemical compounds or other desired products,can then be subjected to a continuous product separations procedure,with or without removing cells and cell debris, and can be performed bycontinuous separations methods well known in the art to separate organicproducts from dilute aqueous solutions and distillation and/orpurifications methods such as those exemplified above and well known inthe art.

In certain embodiments, the fumaric acid producing organisms of theinvention can be sustained, cultured or fermented under anaerobic orsubstantially anaerobic conditions. Briefly, anaerobic conditions refersto an environment devoid of oxygen. Substantially anaerobic conditionsinclude, for example, a culture, batch fermentation or continuousfermentation such that the dissolved oxygen concentration in the mediumremains between 0 and 10% of saturation. Substantially anaerobicconditions also includes growing or resting cells in liquid medium or onsolid agar inside a sealed chamber maintained with an atmosphere of lessthan 1% oxygen. The percent of oxygen can be maintained by, for example,sparging the culture with an N₂/CO₂ mixture.

During or following bioproduction of fumaric acid, a variety of reactionconditions well known in the art can be employed for thecross-metathesis transformation and/or esterification reactions,including diesterification reactions. One particularly useful method forcross-metathesis of fumaric acid to yield two moles of acrylic acid permole of substrate is exemplified in the Examples below. This method issimilarly applicable to the cross-metathesis transformation of fumaratemonoesters and fumarate diesters to yield acrylate esters as describedpreviously. This method as well as any of those cross-metathesisreactions exemplified previously can be employed in conjunction with thebioproduction of fumaric acid to integrate the production of the acrylicacid and/or acrylate esters of the invention.

Similarly, any of a variety of esterification reactions also can beemployed in the conversion of fumaric acid to fumarate monoester orfumarate diester. A particularly useful esterification method also isexemplified further below in the Examples. In certain embodiments,fumaric acid produced by culture or fermentation, as exemplified in FIG.3, can be reacted with alcohols such as ethanol, butanol or any of thosedescribed previously to yield the diesters of fumaric acid, whichsubsequently provide the substrate for cross-metathesis transformationby reaction with ethylene to produce acrylate esters (see, for example,FIG. 2). Formation of fumarate diesters can facilitate separation fromthe aqueous culture medium or fermentation broth and thereby facilitatemetathesis transformation and subsequent isolation of pure acrylateester product. In an embodiment described further below where thebiological production of substrate is derived from renewable feedstocks,production of alcohols such as ethanol and butanol also can be generatedby microbial organisms from renewable feedstocks. Further integration ofalcohol bioproduction from renewable feedstocks can result in all butone carbon of the acrylate esters of the invention being derived fromnon-depletive sources.

Integration of cross-metathesis and/or esterification can be performedusing a variety of process configurations. For example, cross-metathesistransformation can be performed directly in the culture medium and/orfermentation broth. In this embodiment, cross-metathesis and/oresterification regents can be added directly to the medium or broth inconcentrations sufficient to catalyze transformation or esterificationof fumaric acid to acrylic acid or acrylate ester. Similarly, followingesterification or diesterification, esterification reagents canoptionally be removed or neutralized and cross-metathesis reagents canbe added to the medium or broth in concentrations sufficient to catalyzethe transformation of fumarate monoester or fumarate diester to yield anacrylic acid/acrylate ester mixture or to yield acrylate ester.

In a further embodiment, cross-metathesis and/or esterificationreactions also can be performed following any of a variety of treatmentsto the culture medium and/or fermentation broth. For example, the mediumor broth can be treated to adjust the pH, temperature and/or othercharacteristics to a desired level prior to or simultaneously withaddition cross-metathesis and/or esterification reagents. Cells or otherparticulate matter can be removed or partially removed prior to additionof synthesis reagents by, for example, sedimentation, filtration,centrifugation or other method well known in the art. Polypeptidesand/or other soluble macromolecules in the medium and/or broth can beremoved by, for example, precipitation, size exclusion chromatography,ion exchange chromatography or other methods well known in the art. Themedium or broth also can be exchanged or partially exchanged with adesired solution, buffer or reaction formulation suitable or optimal forcross-metathesis transformation and/or esterification. Given theteachings and guidance provided herein, those skilled in the art willknow, or can determine, suitable conditions for couplingcross-metathesis transformation and/or esterification directly in aculture medium or fermentation broth of fumaric acid producing cells.For example, streamlined production of acrylic acid or acrylate esterscan be achieved by coupling the bioproduction and chemical synthesissteps with little to no manipulations of the medium or broth. Yields ofacrylic acid or acrylate ester can be optimized by employing some or allof the above process configurations in conjunction with or prior tocross-metathesis or esterification reactions.

In an alternative embodiment, fumaric acid can be harvested or isolatedat any time point during culture or during the continuous and/ornear-continuous culture period exemplified above and then subjected tocross-metathesis transformation or diesterification followed bycross-metathesis transformation to produce acrylic acid and acrylateester respectively. Those skilled in the art will understand that thelonger the microbial organisms are maintained in a continuous and/ornear-continuous growth phase, the proportionally greater amount offumaric acid can be produced. A variety of purification methods foracrylic acid or acrylate esters are well known in the art. Any of suchmethods can be used for isolation and/or purification of acrylic acid oracrylate ester of the invention.

Therefore, the invention also provides a process for producing anacrylate ester. The process includes: (a) culturing in a sufficientamount of nutrients and media a non-naturally occurring microbialorganism having a set of metabolic modifications obligatorily couplingfumaric acid production to growth of the microbial organism, the set ofmetabolic modifications includes disruption of at least one of the genesets having: (1) fumABC, zwf, purU, or (2) fumABC, zwf, glyA, or anortholog thereof, to produce stable growth-coupled production of fumaricacid; (b) performing diesterification of the fumaric acid to producefumarate diester, and (c) contacting the fumarate diester with asufficient amount of ethylene in the presence of a cross-metathesiscatalyst to produce about two moles of an acrylate ester per mole offumarate diester.

In addition to producing acrylic acid and/or acrylate esters asexemplified in FIG. 3 using glucose as a carbon source for glycolysis,the integrated process of the invention also can be employed to producethese products from renewable feedstocks. Many different carbonsubstrates, such as glucose, sucrose, xylose, arabinose, sorbitol,sucrose, glycerol or synthesis gas (a mixture carbon monoxide, hydrogenand carbon dioxide), can be derived from renewable feedstocks andthereby serve as energy sources for a culture or fermentation process.These and other substrates known in the art can be used for biologicalproduction of fumaric acid.

In some embodiments of the invention, carbon sources for biologicalgrowth and metabolism can be derived from a variety of differentbiomasses. Given the teachings and guidance provided herein, thoseskilled in the art will understand that a fumaric acid or other fumaricacid substrate producing bioprocess of the invention can encompass theuse of a wide range of different carbon sources. Therefore, thebioproduction of substrate such as fumaric acid and/or an alcohol isapplicable for use with a wide range of different carbon sources and/orcarbon source mixtures including, for example, biomass and renewablefeedstocks.

Carbon sources useful for bioproduction of a substrate such as fumaricacid include, for example, sugars or mixtures of sugars or other energysources in growth media, fermentation broth or the like. For example, afumaric acid substrate producing bioprocess of the invention can begenerated where the fumaric acid producing microbial organisms grow onsingle or multiple carbon sources such as on glucose or both on glucoseand arabinose, for example. A culture media can be obtained, produced orsupplemented to contain either or both of these sugars as well as othersugars or carbon sources known in the art. Alternatively, heterogeneousmixtures having or capable of generating the requisite mixtures ofenergy sources also can be used as substrate mixture. A particularexample of such a heterogeneous mixture includes a feedstock including,for example, renewable feedstocks and/or renewable feedstocks derivedfrom biomass. Therefore, carbon source mixtures can include growthmedia, fermentation broth and/or complex feedstocks having more than onedifferent energy source can be used for culture or fermentation of themicrobial organisms of the invention. Other sources of carbon well knownin the art also can be utilized with bioprocess of the invention.

Energy sources within a simple or complex mixture include, for example,carbohydrate, protein, lipid, fat and other macromolecules or chemicalcompounds applicable for conversion by cellular biochemical processes.Such energy sources typically supply the requisite carbon source forenergy production used in biochemical process. Exemplary carbohydratesinclude, for example, simple and complex carbohydrates such asmonosaccharides such as sugars and polysaccharides such as starches,respectively. Exemplary proteins include, for example, all types ofpolypeptides, including proteoglycans. These exemplary macromolecules aswell as lipids, fats and other macromolecules are well known in the artand are all available as energy sources for the sets of complementarymetabolizing organisms of the invention.

Exemplary materials and/or substances supplying these energy sourceswithin complex mixtures such as biomass and/or renewable feedstocksinclude, for example, those described previously as well as otherrenewable resources or byproducts well known to those skilled in theart. For example, biomass can provide a wide variety of energy sourcesincluding the above carbohydrate, protein, lipid, fat as well as othermolecules such as aromatic compounds and/or proteineaceous substancessuch as lignin. Biomass and renewable feedstocks are particularly usefulas sources of a variety of carbohydrate. Such sources include, forexample, cellulosic biomass, a hemicellulosic biomass, wheat straw, cornstover, reed canary grass, starch, corn, wheat or cotton woodchipsstarch, corn, wheat, cotton. Portions, chaff, fractions and wasteproducts, for example, of these exemplary biomasses and renewablefeedstocks as well as others well known in the art also are particularlyuseful sources for a variety of carbohydrates that can be used in agrowth medium for a set of complementary metabolizing organisms of theinvention. Particularly useful carbon sources include medium orfeedstocks containing different simple or complex carbohydrates.Carbohydrates provide an efficient carbon source for cellularproliferation. Exemplary carbohydrates include the sugars glucose,sucrose, xylose, arabinose, galactose, mannose or fructose.

Feedstocks containing the sugar energy sources exemplified above orother carbon sources useful for growth of the complementary metabolizingorganisms of the invention include, for example, cellulosic biomass,hemicellulosic biomass and lignin feedstocks. Such biomass feedstockscontain, for example, carbohydrate substrates useful as carbon sourcessuch as glucose, sucrose, xylose, arabinose, galactose, mannose,fructose and starch.

In other embodiments, hydrolysis of biomass can generate toxic compoundswhich also can be beneficially utilized from the substrate media ascarbon sources for bioprocessing. Exemplary toxic compounds that can beharnessed as carbon or other fuel sources include furfurals, aromatics,acetate and other undetermined substrates. Removal of these toxiccompounds also is particularly useful to the overall cost effectivenessof the process because it eliminates requirements for implementation ofseparate unit operations prior to, for example, the actual bioconversionstep. When used as a carbon source, toxic compounds can be consumed, forexample, before the main bioconversion takes place or concurrently inthe same reaction vessel. One specific embodiment, achieves toxicproduct removal by conversion into cell matter or other products ofinterest.

Briefly, microbial organisms can be designed and generated to utilizeone or more byproducts, including toxic byproducts, generated duringco-culture of the complementary metabolizing organisms. For example, asubstrate producing microbial organism also can be modified tometabolize a byproduct of the culture or fermentation itself. In thisspecific embodiment, the initial carbon source contained in a mediumsupporting growth and metabolism produces a renewable energy source thatis further utilized by, for example, the modified organism.

Any of the integrated processes of the invention described above can beconfigured as a production system useful for the manufacture of acrylicacid and/or acrylate esters. The amounts of acrylic acid or acrylateester that can be manufactured can range from small, research quantitiesto large commercial-scale amounts. In the former, those skilled in theart will understand that small cultures of fumaric acid producingorganisms can be useful for ease of handling and efficiency. In thelatter, those skilled in the art will understand that fermentation-sizecultures of fumaric acid producing organisms can be useful toefficiently achieve desired productivity levels.

A production system of the invention can be configured in a variety ofdifferent ways. For example, a production system can contain some or allof the components needed to generate fumaric acid, acrylic acid and/oracrylate ester. In the specific embodiment where the production systemcontains all of the components, the fumaric acid producing cells can bein stationary or log growth phase. A production system also can containless than all components and be poised for cell growth, fumaric acidproduction, acrylic acid production and/or acrylate ester production bythe addition of one or more components of the previously describedintegrated process of the invention.

Therefore, the invention further provides acrylic acid productionsystem. The production system includes: (a) a culture of a non-naturallyoccurring microbial organism having a set of metabolic modificationsobligatorily coupling fumaric acid production to growth of the microbialorganism, the set of metabolic modifications includes disruption of atleast one of the gene sets having: (1) fumABC, zwf, purU, or (2) fumABC,zwf, glyA, or an ortholog thereof, which confer stable growth-coupledproduction of fumaric acid, and (b) an amount of ethylene and across-metathesis transformation catalyst sufficient to produce about twomoles of acrylic acid per mole of fumaric acid.

An acrylate ester production system is also provided. The productionsystem includes: (a) a culture of a non-naturally occurring microbialorganism having a set of metabolic modifications obligatorily couplingfumaric acid production to growth of the microbial organism, the set ofmetabolic modifications includes disruption of at least one of the genesets having: (1) fumABC, zwf, purU, or (2) fumABC, zwf, glyA or anortholog thereof, which confer stable growth-coupled production offumaric acid; (b) at least one diesterification reagent sufficient toproduce fumarate diester from the fumaric acid, and (c) an amount ofethylene and a cross-metathesis catalyst sufficient to produce about twomoles of an acrylate ester per mole of fumarate diester.

It is understood that modifications which do not substantially affectthe activity of the various embodiments of this invention are alsoincluded within the definition of the invention provided herein.Accordingly, the following examples are intended to illustrate but notlimit the present invention.

EXAMPLE I

This Example describes chemical synthesis methods for cross-metathesisof fumaric acid to acrylic acid and esters thereof and for thediesterification of fumaric acid to fumarate diester.

Acrylic acid from fumaric acid and ethylene: Briefly, a 1 L glassreactor composed of thick wall glass is charged under nitrogen or argonwith an appropriate solvent such as dichloromethane or dichloroethane(500 mL), fumaric acid (100 g, 0.86 mol), and the Grubbs Rutheniummetathesis catalyst (1.0-0.01 mol %). After stirring for 10-60 min undernitrogen, the vessel is pressurized with 1.0-5.0 atm of ethylene gas andthe reaction is stirred at 0-50° C. over a period of up to 24 hours oruntil process monitoring indicates the reaction is complete. The unusedethylene is then removed and recovered and the reaction vessel is openedto the atmosphere. The solution is treated with aqueous sodium hydroxide(300-500 mL, 1-5 M solution) and the aqueous layer is extracted twicewith the above solvent. The aqueous layer is then acidified to pH 0-2and extracted with dichloromethane or diethylether (5×100 mL). Followingremoval of the solvent, hydroquinone is added to limit polymerization,and the crude acrylic acid is purified by distillation (b.p. 139-140°C.).

Dialkyl esters of fumaric acid: Dialkylfumarate esters or the diestersof fumaric acid

(e.g., dimethyl and dibutyl fumarate) are readily available from manycommercial sources and are prepared by various routes includingdiesterification of fumaric acid with aliphatic alcohols in the presenceof a p-toluene sulfonic acid catalyst. Alternatively, the esters can beprepared from fumaryl chloride and alkyl alcohols using an aminecatalyst. A representative example is provided below.

Fisher Synthesis of Dialkyl Fumarate Esters is performed as describedin, for example, U.S. patent application 20020040123 A1. Briefly,monomer synthesis from fumaric acid and 1-eicosanol is performed byadding into the reaction flask (equipped with a condenser and aDean-Stark trap apparatus to remove the reaction water as it formed),2.8 g (FW 116.07, 0.01875 moles) of fumaric acid, 11.2 g (0.0375 moles)of 1-eicosanol (FW 298.56), 0.3567 g (0.00188 mole) of.rho.-toluenesulfonic acid monohydrate, and 50 mL to toluene. Themixture was heated at 130.degree. C. for 18 hours under nitrogen. Thereaction was then cooled to room temperature and filtered and solventtoluene was removed by a rotary evaporator to obtain the product (mp71-73.degree. C.). The C.sub.20 fumarate ester product was characterizedby IR and NMR spectroscopy. The IR spectrum of the product was recordedas the melted solid film in NaCl plates. The spectrum showed an esterpeak at 1728 cm.sup.-1 and a double bond absorption peak at 1647cm.sup.-1. sup. 13C NMR of the product showed the double bond absorptionpeak at 134.0 ppm (trans —HC.dbd.CH—, carbon) and the carbonyl esterpeak at 165 ppm. The NMR spectrum also showed an absorption peak at 66ppm due to a methylene next to ester functionality (—C(O)O—CH.sub.2-).The absorption peaks in the aliphatic region are typical of the straightchain alkyl groups.

Alkyl acrylate esters from dialkyl fumarate and ethylene: The samegeneral protocol is employed as described above with the reaction vesselbeing charged with dialkyl fumarate rather than fumaric acid. The finalmixture following completion of the reaction would be processed bycrystallization or distillation to obtain the purified alkyl acrylate.

EXAMPLE II

This Example describes the combined biosynthesis and chemical of acrylicacid.

Acrylic Acid from biologically produced fumaric acid: Acrylic acid willbe produced by reaction between fumaric acid produced by fermentationand ethylene in the presence of a suitable catalyst (e.g., Grubbscatalyst). In this case, a fermentation process is implemented using anorganism engineered for high level production of fumaric acid.Performing the metathesis process directly on the fermentation brothfollowing completion of the fermentation process is the preferredprocess. A general procedure for the combined fermentation andmetathesis process is as follows:

The production organism is grown in a 10 L bioreactor sparged with anN2/CO2 mixture, using 5 L broth containing 5 g/L potassium phosphate,2.5 g/L ammonium chloride, 0.5 g/L magnesium sulfate, and 30 g/L cornsteep liquor, and an initial glucose concentration of 20 g/L. As thecells grow and utilize the glucose, additional 70% glucose is fed intothe bioreactor at a rate approximately balancing glucose consumption.The temperature of the bioreactor is maintained at 30 degrees C. Growthcontinues for approximately 24 hours, until fumaric acid reaches aconcentration of between 10-200 g/L, with the cell density being between5 and 50 g/L. Upon completion of the cultivation period, the fermentercontents are passed through a cell separation unit (e.g., centrifuge) toremove cells and cell debris, and the fermentation broth is transferredto a secondary reaction unit where Grubbs catalyst (1.0-0.01 mol %) isadded to the broth, possibly along with an appropriate organic solventto increase catalyst solubility, and the reactor is pressurized withethylene (1.0-5.0 atm). After stirring the time required for completereaction, ethylene pressure is released and recovered, and acrylic acidis separated from the broth and purified as described above.

EXAMPLE III

This example demonstrates the conversion of diethylfumarate to ethylacrylate.

Example of metathesis of fumarate: In order to demonstrate thefeasibility of converting fumarate(s) to acrylate(s) through theaddition of ethylene, a series of commercially available metathesiscatalysts were screened. The following results demonstrate the abilityof the metathesis reaction to take place and suggest areas to explorefor enhanced performance.

General: Experiments were conducted in 150-mL Fisher-Porter pressurebottles at 150 psi. Compressed ethylene (99.95%) was purchased fromPraxair and used as received. All solvents and chemicals were purchasedfrom Aldrich Chemicals. Diethyl fumarate (98%) was distilled before use.Diethyl maleate, dimethyl fumarate, ethyl acrylate, and acrylic acidwere used as received. All catalysts were prepared by Materia, Inc. andobtained from either Materia or Aldrich Chemicals. All GasChromatography (GC) data were acquired with Agilent Technologies 6850Series II using the HP-5 column of J&W Scientific. The temperatureprofile was held at 100° C. for 1 minute, ramped up to 250° C. with therate of 10° C. per minute, and held at 250° C. for 5 minutes. Nuclearmagnetic resonance (NMR) data was obtained from the Varian 400 MHzinstrument. NMR solvents were purchased from Cambridge Isotope Inc.

Standard Procedure: Substrate (e.g., diethylfumarate, 5 g), catalyst(2.5 mol %) and magnetic stirring bar were added to a Fisher-Porterbottle inside a nitrogen-filled glove box. The Fisher-Porter bottle wasassembled and the apparatus was moved out of the box and connected to anethylene cylinder. The ethylene line was purged with ethylene for aseveral minutes and the Fisher-Porter bottle was then pressurized withethylene to the desired level (150 psi). The bottle was placed into anoil bath at 60° C. on the hot plate of a magnetic stirrer. After theindicated reaction time, the bottle was removed from the oil bath andcooled to room temperature in air. The pressure was released and thecontents were filtered through filter paper. An aliquot was diluted indichloromethane or chlorobenzene in a GC vial and the sample wasanalyzed by GC for percent conversion of diethyl fumarate to ethylacrylate.

The catalysts screened in this experiment included five commerciallyavailable catalysts. All catalysts can be obtained from Materia or fromSigma-Aldrich. Full details on these five catalysts are provided in theTable 4 below.

TABLE 4 List of commercially available catalysts screened (Table takenfrom the Materia, Inc. product catalog located on the world wide web atmateria-inc.com) Sigma-Aldrich Chemical Structure Product & CAS #Product Description Product #

C627 [301224-40-8] Hoveyda-Grubbs Second Generation CatalystC₃₁H₃₈Cl₂N₂ORu Ruthenium, [1,3-bis(2,4,6-trimethylphenyl)-2-imidazolidinylidene]dichloro[[2- (1-methylethoxy)phenyl]methylene]   FW  626.62 569755

C793 [927429-60-5] C₄₂H₅₇Cl₂N₂PRu [1,3-Bis(2-methylphenyl)-2-imidazolidinylidene]dichloro(benzylidene)(tricyclohexylphosphine)ruthenium(II) FW 792.87 682284

C823 [172222-30-9] Grubbs First Generation Catalyst C₄₃H₇₂Cl₂P₂RuRuthenium, dichloro(phenylmethylene)bis (tricyclohexylphosphine)   FW  822.95 579726

C827 [253688-91-4] C₄₄H₆₇Cl₂N₂PRu [1,3-Bis(2,4,6-trimethylphenyl)-2-imidazolidinylidene]dichloro(3- methyl-2-butenylidene)(tricyclohexylphosphine)ruthenium(II) FW 826.97 682365

C848 [246047-72-3] Grubbs Second Generation Catalyst C₄₆H₆₅Cl₂N₂PRuRuthenium, [1,3-bis-(2,4,6-trimethylphenyl)-2-imidazolidinylidene]dichloro(phenylmethylene) (tricyclohexylphosphine)  FW   848.97 569747

Screening the catalysts shown in Table 4 under the experimentalconditions described above yielded results demonstrating the conversionof the fumarate(s) into acrylate(s). The results for diethyl fumarateare shown in the Table 5 below and in FIG. 4. These reactions wereperformed with neat substrate and no additional solvent. The reactionsgenerally proceeded in a sluggish manner, especially with Generation 1and 2 Grubbs catalysts, where no conversion (C823) and 1% conversion(C848) were observed, respectively. The best conversion and yield of 7%was seen with catalyst C627, which is the only phosphine-free systemtested. Similar results were achieved with dimethyl fumarate.

TABLE 5 Results of catalyst screening (EA: ethyl acrylate, DEF: diethylfumarate; IE: itaconic acid diethyl ester) Cat C2H4 T t Composition(Area %) Catalyst (mol %) (psi) (C.) (h) EA DEF IE C627 2.5 150 60 4 788 4 C793 2.5 150 60 4 1 96 0 C823 2.5 150 60 16 0 93 0 C827 2.5 150 6016 1 93 0 C848 2.5 150 60 4 1 93 0

EXAMPLE IV

This example demonstrates the biosynthesis of fumaric acid.

Example of biosynthesis of fumaric acid: Escherichia coli K-12 MG1655served as the wild-type strain into which the deletions are introduced.Deletions of E. coli genes fumABC, zwf and purU was performed by usingthe well-known Red E/T technology. The strains were constructed byincorporating in-frame deletions using homologous recombination via theλ Red recombinase system of Datsenko and Wanner. The approach involvedreplacing a chromosomal sequence (i.e., the gene targeted for removal)with a selectable antibiotic resistance gene, which itself was laterremoved. No drug resistance markers remained after each deletion,allowing accumulation of multiple mutations in each target strain.

Production of fumarate. Wild type E. coli, strain 1 (ΔfumABC, Δzwf) andstrain 2 (ΔfumABC, Δzwf, ΔpurU) were tested in shake flask culturesbefore subjecting them to adaptive evolution. Cultures were grownaerobically in M9 minimal medium containing 2 g/L glucose, andconcentrations of glucose, fumarate, and other organic acid products inthe culture supernatant were determined by HPLC using an HPX-87H column(BioRad). While the wild-type E. coli MG1655 did not secrete anyfumarate, strain 1 secreted 0.1 mol fumarate per mol of glucose consumedover 48 h. No other byproducts were detected from the HPLC measurements.Quite surprisingly, strain 2 that has an additional deletion in purUformed slightly more fumarate (0.125±0.014 mol/mol glucose consumed) anda lot of acetate (0.90 mol/mol glucose consumed).

After briefly evolving strain 2 in chemostat for 8 days, it was observedthat the growth rate improved for 0.38 per hour to 0.48 per hour. Thisis in reasonable agreement with the growth rate of 0.58 per hourpredicted by our models. However, the measured fumarate yield did notsignificantly increase in shake flask cultures.

TABLE 1 Reaction combinations targeted for removal to enhance succinateproduction in E. coli.. 1. FUM 2. FUM MTHFC PGDH 3. FUM MTHFC PGL 4.FTHFD FUM G6PDHy 5. FUM G6PDHy MTHFC 6. FTHFD FUM PGL 7. FUM G6PDHyGLYCL 8. FUM GLYCL PGDH 9. FUM GLYCL PGL 10. FTHFD FUM TKT1 11. FUMMTHFC TKT1 12. FUM MTHFC TAL 13. FTHFD FUM TAL 14. FUM GLYCL TKT1 15.FUM GLYCL TAL 16. FUM MTHFC RPE 17. FTHFD FUM RPE 18. FUM GLYCL RPE 19.FUM MTHFC TKT2 20. FTHFD FUM TKT2 21. FUM GLYCL TKT2 22. MDH ME1x ME223. GLYCL NADH6 PGI 24. FUM G6PDHy GLUDy MTHFC 25. FTHFD FUM G6PDHyGLUDy 26. FDH2 FUM GLUDy PGL 27. FUM GLUDy MTHFC PGDH 28. FDH2 FUMG6PDHy GLUDy 29. FDH2 FUM GLUDy PGDH 30. FUM GLUDy MTHFC PGL 31. FTHFDFUM GLUDy PGL 32. FUM GLUDy GLYCL PGDH 33. FUM G6PDHy GLUDy GLYCL 34.FUM GLUDy GLYCL PGL 35. FDH2 FUM GLUDy TKT1 36. FDH2 FUM GLUDy TAL 37.FTHFD FUM GLUDy TAL 38. FTHFD FUM GLUDy TKT1 39. FUM GLUDy MTHFC TKT140. FUM GLUDy MTHFC TAL 41. FUM GLUDy GLYCL TKT1 42. FUM GLUDy GLYCL TAL43. FUM G6PDHy MTHFC THD2 44. FUM MTHFC PGL THD2 45. FUM MTHFC PGDH THD246. FTHFD FUM G6PDHy THD2 47. FTHFD FUM PGL THD2 48. FUM GLYCL PGDH THD249. FUM G6PDHy GLYCL THD2 50. FUM GLYCL PGL THD2 51. FDH2 FUM GLUDy RPE52. FUM GLUDy MTHFC RPE 53. FTHFD FUM GLUDy RPE 54. FUM GLUDy GLYCL RPE55. FUM MTHFC PDH PGDH 56. FTHFD FUM PDH PGL 57. FTHFD FUM G6PDHy PDH58. FUM MTHFC PDH PGL 59. FTHFD FUM PDH PGDH 60. FUM G6PDHy MTHFC PDH61. FUM GLYCL PDH PGDH 62. FUM GLYCL PDH PGL 63. FUM G6PDHy GLYCL PDH64. FUM GLCpts MTHFC PGDH 65. FUM G6PDHy GLCpts MTHFC 66. FTHFD FUMG6PDHy GLCpts 67. FTHFD FUM GLCpts PGL 68. FTHFD FUM GLCpts PGDH 69. FUMGLCpts MTHFC PGL 70. FUM GLCpts GLYCL PGDH 71. FUM G6PDHy GLCpts GLYCL72. FUM GLCpts GLYCL PGL 73. FDH2 FUM GLUDy TKT2 74. FTHFD FUM GLUDyTKT2 75. FUM GLUDy MTHFC TKT2 76. FUM GLUDy GLYCL TKT2 77. FUM MTHFC TALTHD2 78. FUM MTHFC THD2 TKT1 79. FTHFD FUM TAL THD2 80. FTHFD FUM THD2TKT1 81. FUM GLYCL TAL THD2 82. FUM GLYCL THD2 TKT1 83. FTHFD FUM PDHTKT1 84. FTHFD FUM PDH TAL 85. FUM MTHFC PDH TAL 86. FUM MTHFC PDH TKT187. FUM GLYCL PDH TAL 88. FUM GLYCL PDH TKT1 89. FUM GLCpts MTHFC TAL90. FTHFD FUM GLCpts TKT1 91. FTHFD FUM GLCpts TAL 92. FUM GLCpts MTHFCTKT1 93. FUM GLCpts GLYCL TAL 94. FUM GLCpts GLYCL TKT1 95. CBMK2 FTHFDFUM PGDH 96. CBMK2 FUM MTHFC PGL 97. CBMK2 FUM MTHFC PGDH 98. CBMK2 FUMG6PDHy MTHFC 99. CBMK2 FTHFD FUM PGL 100. CBMK2 FTHFD FUM G6PDHy 101.FTHFD FUM RPE THD2 102. FUM MTHFC RPE THD2 103. CBMK2 FUM G6PDHy GLYCL104. CBMK2 FUM GLYCL PGL 105. CBMK2 FUM GLYCL PGDH 106. FUM GLYCL RPETHD2 107. FUM G6PDHy GLU5K MTHFC 108. FUM G5SD MTHFC PGDH 109. FUM G5SDMTHFC PGL 110. FUM G5SD G6PDHy MTHFC 111. FTHFD FUM G5SD PGL 112. FUMGLU5K MTHFC PGL 113. FTHFD FUM G5SD PGDH 114. FTHFD FUM GLU5K PGL 115.FTHFD FUM G5SD G6PDHy 116. FTHFD FUM GLU5K PGDH 117. FTHFD FUM G6PDHyGLU5K 118. FUM GLU5K MTHFC PGDH 119. ASNS2 FUM G6PDHy MTHFC 120. ASNS2FTHFD FUM PGL 121. ASNS2 FUM MTHFC PGL 122. ASNS2 FTHFD FUM PGDH 123.ASNS2 FUM MTHFC PGDH 124. ASNS2 FTHFD FUM G6PDHy 125. FUM GLU5K GLYCLPGDH 126. FUM G5SD GLYCL PGL 127. FUM G5SD G6PDHy GLYCL 128. FUM GLU5KGLYCL PGL 129. FUM G5SD GLYCL PGDH 130. FUM G6PDHy GLU5K GLYCL 131.ASNS2 FUM GLYCL PGL 132. ASNS2 FUM G6PDHy GLYCL 133. ASNS2 FUM GLYCLPGDH 134. FDH2 FORt FUM PGDH 135. FDH2 FORt FUM PGL 136. FDH2 FORt FUMG6PDHy 137. FUM MTHFC PDH RPE 138. FTHFD FUM PDH RPE 139. FUM GLYCL PDHRPE 140. FUM GLCpts MTHFC RPE 141. FTHFD FUM GLCpts RPE 142. FUM GLCptsGLYCL RPE 143. CBMK2 FUM MTHFC TKT1 144. CBMK2 FTHFD FUM TAL 145. CBMK2FTHFD FUM TKT1 146. CBMK2 FUM MTHFC TAL 147. CBMK2 FUM GLYCL TAL 148.CBMK2 FUM GLYCL TKT1 149. FTHFD FUM THD2 TKT2 150. FUM MTHFC THD2 TKT2151. FUM G5SD MTHFC TKT1 152. FTHFD FUM G5SD TKT1 153. FTHFD FUM G5SDTAL 154. FTHFD FUM GLU5K TKT1 155. FUM GLU5K MTHFC TAL 156. FTHFD FUMGLU5K TAL 157. FUM G5SD MTHFC TAL 158. FUM GLU5K MTHFC TKT1 159. ASNS2FTHFD FUM TKT1 160. ASNS2 FUM MTHFC TAL 161. ASNS2 FTHFD FUM TAL 162.ASNS2 FUM MTHFC TKT1 163. FUM GLYCL THD2 TKT2 164. FUM GLU5K GLYCL TKT1165. FUM G5SD GLYCL TKT1 166. FUM GLU5K GLYCL TAL 167. FUM G5SD GLYCLTAL 168. ASNS2 FUM GLYCL TKT1 169. ASNS2 FUM GLYCL TAL 170. FTHFD FUMPDH TKT2 171. FUM MTHFC PDH TKT2 172. FDH2 FORt FUM TAL 173. FDH2 FORtFUM TKT1 174. FUM GLYCL PDH TKT2 175. FTHFD FUM GLCpts TKT2 176. FUMGLCpts MTHFC TKT2 177. FUM GLCpts GLYCL TKT2 178. CBMK2 FUM MTHFC RPE179. CBMK2 FTHFD FUM RPE 180. CBMK2 FUM GLYCL RPE 181. FUM GLU5K MTHFCRPE 182. FUM G5SD MTHFC RPE 183. FTHFD FUM GLU5K RPE 184. FTHFD FUM G5SDRPE 185. ASNS2 FUM MTHFC RPE 186. ASNS2 FTHFD FUM RPE 187. FUM G5SDGLYCL RPE 188. FUM GLU5K GLYCL RPE 189. ASNS2 FUM GLYCL RPE 190. FDH2FORt FUM RPE 191. CBMK2 FTHFD FUM TKT2 192. CBMK2 FUM MTHFC TKT2 193.CBMK2 FUM GLYCL TKT2 194. FUM GLU5K MTHFC TKT2 195. FTHFD FUM G5SD TKT2196. FTHFD FUM GLU5K TKT2 197. FUM G5SD MTHFC TKT2 198. ASNS2 FUM MTHFCTKT2 199. ASNS2 FTHFD FUM TKT2 200. FUM GLU5K GLYCL TKT2 201. FUM G5SDGLYCL TKT2 202. ASNS2 FUM GLYCL TKT2 203. FDH2 FORt FUM TKT2 204. ACt6FUM MTHFC THD5 205. ACt6 FDH2 FUM THD5 206. ACt6 FTHFD FUM THD5 207.ACt6 FUM GLYCL THD5 208. FDH2 FUM PTAr THD5 209. FUM HEX1 PGI PPS 210.ACKr FTHFD FUM THD5 211. ACKr FDH2 FUM THD5 212. FTHFD FUM PTAr THD5213. FUM GLCt2 PGI PPS 214. FUM MTHFC PTAr THD5 215. ACKr FUM MTHFC THD5216. FUM GLYCL PTAr THD5 217. ACKr FUM GLYCL THD5 218. ACt6 FUM HEX1 PPS219. ACt6 FUM GLCt2 PPS 220. FUM HEX1 PPS PTAr 221. FUM GLCt2 PPS PTAr222. ACKr FUM HEX1 PPS 223. ACKr FUM GLCt2 PPS 224. FUM MTHFC PDH PGI225. FTHFD FUM PDH PGI 226. FUM GLYCL PDH PGI 227. ACt6 FUM MTHFC PGI228. ACt6 FTHFD FUM PGI 229. ACt6 FUM GLYCL PGI 230. MDH ME1x ME2 SUCOAS231. FUM MTHFC PGI PTAr 232. FTHFD FUM PGI PTAr 233. ACKr FTHFD FUM PGI234. FUM GLYCL PGI PTAr 235. ACKr FUM MTHFC PGI 236. HEX1 MDH PGI PPS237. ACKr FUM GLYCL PGI 238. GLCt2 MDH PGI PPS 239. PDH PGDH PPS THD2240. FUM MTHFC PGL PGM THD2 241. ENO FUM G6PDHy MTHFC THD2 242. ENOFTHFD FUM PGL THD2 243. ENO FTHFD FUM G6PDHy THD2 244. ENO FUM MTHFC PGLTHD2

TABLE 2 A list of all the reaction stoichiometries and the associatedgenes known to be associated with the reactions identified for deletionin the strategies listed in Table 1. Reaction Abbreviation ReactionStoichiometry Associated genes ACKr [c]: ac + atp <==> actp + adp b2296,b3115 ACt6 ac[e] + h[e] <==> ac[c] + h[c] b4067 ASNS2 [c]: asp-L + atp +nh4 --> amp + asn-L + h + ppi b3744 CBMK2 [c]: atp + co2 + nh4 --> adp +cbp + (2) h b0323, b2874, b0521 ENO [c]: 2pg <==> h2o + pep b2779 FDH2for[c] + (3) h[c] + ubq8[c] --> co2[c] + (2) h[e] + b3893 + b3893 +b3894, b4079, b1474 + b1475 + b1476 ubq8h2[c] FORt for[e] <==> for[c]b0904, b2492 FTHFD [c]: 10fthf + h2o --> for + h + thf b1232 FUM [c]:fum + h2o <==> mal-L b1611, b1612, b4122 G5SD [c]: glu5p + h + nadph -->glu5sa + nadp + pi b0243 G6PDHy [c]: g6p + nadp <==> 6pgl + h + nadphb1852 GLCpts glc-D[e] + pep[c] --> g6p[c] + pyr[c] b2417, b1101, b2415,b2416, b2417, b1621, b2415, b2416, b1817, b1818, b1819, b2415, b2416GLCt2 glc-D[e] + h[e] --> glc-D[c] + h[c] b2943 GLU5K [c]: atp + glu-L--> adp + glu5p b0242 GLUDy [c]: glu-L + h2o + nadp <==> akg + h +nadph + nh4 b1761 GLYCL [c]: 10fthf + h2o --> for + h + thf b1232 HEX1[c]: atp + glc-D --> adp + g6p + h b2388 MDH [c]: mal-L + nad <==> h +nadh + oaa b3236 ME1x [c]: mal-L + nad --> co2 + nadh + pyr b1479 ME2[c]: mal-L + nadp --> co2 + nadph + pyr b2463 MTHFC [c]: h2o + methf<==> 10fthf + h b0529 NADH6 (4.5) h[c] + nadh[c] + ubq8[c] --> (3.5)h[e] + nad[c] + b2276, b2277, b2278, b2279, b2280, b2281, b2282,ubq8h2[c] b2283, b2284, b2285, b2286, b2287, b2288 PDH [c]: coa + nad +pyr --> accoa + co2 + nadh b0114, b0115, b0116 PGDH [c]: 6pgc + nadp -->co2 + nadph + ru5p-D b2029 PGI [c]: g6p <==> f6p b4025 PGL [c]: 6pgl +h2o --> 6pgc + h b0767 PGM [c]: 3pg <==> 2pg b3612 PPS [c]: atp + h2o +pyr --> amp + (2) h + pep + pi b1702 PTAr [c]: accoa + pi <==> actp +coa b2297, b2458 RPE [c]: ru5p-D <==> xu5p-D b3386, b4301 SUCOAS [c]:atp + coa + succ <==> adp + pi + succoa b0728, b0729 TAL [c]: g3p + s7p<==> e4p + f6p b0008, b2464 THD2 (2) h[e] + nadh[c] + nadp[c] --> (2)h[c] + nad[c] + b1602 + b1603 nadph[c] THD5 [c]: nad + nadph --> nadh +nadp b1602 + b1603, b3962 TKT1 [c]: r5p + xu5p-D <==> g3p + s7p b2935,b2465 TKT2 [c]: e4p + xu5p-D <==> f6p + g3p b2935, b2465

TABLE 3 List of the metabolite abbreviations, the corresponding namesand locations of all the metabolites that participate in the reactionslisted in Supplementary Table 2. Metabolite Abbreviation CompartmentMetabolite Name 10fthf Cytosol 10-Formyltetrahydrofolate 13dpg Cytosol3-Phospho-D-glyceroyl phosphate 2dmmq8 Cytosol 2-Demethylmenaquinone 82dmmql8 Cytosol 2-Demethylmenaquinol 8 2h3opp Cytosol2-Hydroxy-3-oxopropanoate 2pg Cytosol D-Glycerate 2-phosphate 3pgCytosol 3-Phospho-D-glycerate 6pgc Cytosol 6-Phospho-D-gluconate 6pglCytosol 6-phospho-D-glucono-1,5-lactone Ac Cytosol Acetate ac[e]Extra-organism Acetate Accoa Cytosol Acetyl-CoA Actp Cytosol Acetylphosphate Adp Cytosol ADP Akg Cytosol 2-Oxoglutarate asn-L CytosolL-asparagine asp-L Cytosol L-aspartate Atp Cytosol ATP Cbp CytosolCarbamoyl phosphate co2 Cytosol CO2 Coa Cytosol Coenzyme A Dha CytosolDihydroxyacetone Dhap Cytosol Dihydroxyacetone phosphate dhor-S Cytosol(S)-Dihydroorotate e4p Cytosol D-Erythrose 4-phosphate Etoh CytosolEthanol etoh[e] Extra-organism Ethanol f6p Cytosol D-Fructose6-phosphate Fad Cytosol FAD fadh2 Cytosol FADH2 Fdp Cytosol D-Fructose1,6-bisphosphate Fgam Cytosol N2-Formyl-N1-(5-phospho-D-ribosyl)glycinamide For Cytosol Formate for[e] Extra-organism FormateFum Cytosol Fumarate fum[e] Extra-organism Fumarate g3p CytosolGlyceraldehyde 3-phosphate g6p Cytosol D-Glucose 6-phosphate Gar CytosolN1-(5-Phospho-D-ribosyl)glycinamide glc-D[e] Extra-organism D-Glucoseglu5p Cytosol L-glutamate 5-phosphate glu5sa Cytosol L-glutamate5-semialdehyde glu-L Cytosol L-Glutamate Glx Cytosol Glyoxylate GlyCytosol Glycine Glyclt Cytosol Glycolate glyclt[e] Extra-organismGlycolate glyc-R Cytosol (R)-Glycerate H Cytosol H+ h[e] Extra-organismH+ h2o Cytosol H2O hom-L Cytosol L-Homoserine lac-D Cytosol D-Lactatelac-D[e] Extra-organism D-Lactate mal-L Cytosol L-Malate Methf Cytosol5,10-Methenyltetrahydrofolate Mlthf Cytosol5,10-Methylenetetrahydrofolate Nad Cytosol Nicotinamide adeninedinucleotide Nadh Cytosol Nicotinamide adenine dinucleotide - reducedNadp Cytosol Nicotinamide adenine dinucleotide phosphate Nadph CytosolNicotinamide adenine dinucleotide phosphate - reduced nh4 CytosolAmmonium o2 Cytosol O2 Oaa Cytosol Oxaloacetate orn-L CytosolL-Ornithine Orot Cytosol Orotate Pep Cytosol Phosphoenolpyruvate PhomCytosol O-Phospho-L-homoserine Pi Cytosol Phosphate pi[e] Extra-organismPhosphate Ppa Cytosol Propionate Ppcoa Cytosol Propanoyl-CoA Ppi CytosolDiphosphate Ptrc Cytosol Putrescine Pyr Cytosol Pyruvate pyr[e]Extra-organism Pyruvate r5p Cytosol alpha-D-Ribose 5-phosphate ru5p-DCytosol D-Ribulose 5-phosphate s7p Cytosol Sedoheptulose 7-phosphateSucc Cytosol Succinate succ[e] Extra-organism Succinate Succoa CytosolSuccinyl-CoA Thf Cytosol 5,6,7,8-Tetrahydrofolate thr-L CytosolL-Threonine ubq8 Cytosol Ubiquinone-8 ubq8h2 Cytosol Ubiquinol-8 xu5p-DCytosol D-Xylulose 5-phosphate

Throughout this application various publications have been referencedwithin parentheses. The disclosures of these publications in theirentireties are hereby incorporated by reference in this application inorder to more fully describe the state of the art to which thisinvention pertains.

Although the invention has been described with reference to thedisclosed embodiments, those skilled in the art will readily appreciatethat the specific examples and studies detailed above are onlyillustrative of the invention. It should be understood that variousmodifications can be made without departing from the spirit of theinvention. Accordingly, the invention is limited only by the followingclaims.

1. A process comprising a) culturing by fermentation in a sufficientamount of nutrients and media a microbial organism that produces fumaricacid; b) esterification of said fumaric acid to provide a fumaratediester; and c) olefin metathesis of said fumarate diester to anacrylate ester in the presence of an olefin metathesis catalyst selectedfrom ruthenium,[1,3-bis(2,4,6-trimethylphenyl)-2-imidazolidinylidene]dichloro[[2-(1-methoxy)phenyl]methylene],[1,3-Bis(2-methylphenyl)-2-imidazolidene]dichloro(benzylidene)(tricyclohexylphosphine) ruthenium (II), ruthenium,dichloro(phenylmethylene)bis(tricyclohexylphospine),[1,3-Bis(2,4,6-trimethylphenyl)-2-imidazolidinylidene]dichloro(3-methyl-2-butenylidene)(tricyclohexylphosphine)ruthenium (II), and ruthenium,[1,3-bis(2,4,6-trimethylphenyl)-2-imidazolidinylidene]dichloro(phenylmethylene)(tricyclohexylphosphine).